Nitric oxide-blocked cross-linked tetrameric hemoglobin

ABSTRACT

The present invention includes compositions containing carboxamidomethylated cross-linked hemoglobin where the cysteine moiety of the hemoglobin includes a thiol protecting group and where the hemoglobin has a reduced ability to bind with nitric oxide. Preferably, the hemoglobin is deoxygenated, endotoxin free, and stroma free. The present invention also includes method of preparation, process of preparation and the method of use including supplementing blood volume in mammals and treating disorders in mammals where oxygen delivery agents are of benefit.

CLAIM OF PRIORITY

The present application is a continuation of, and claims priority toU.S. patent application entitled “NITRIC OXIDE-BLOCKED CROSS-LINKEDTETRAMERIC HEMOGLOBIN,” Ser. No. 12/287,558 filed Oct. 10, 2008, whichclaims priority to U.S. patent application entitled “NITRICOXIDE-BLOCKED CROSS-LINKED TETRAMERIC HEMOGLOBIN,” Ser. No. 11/713,195filed Mar. 1, 2007, now U.S. Pat. No. 7,504,377, which claims priorityto U.S. Provisional Application Ser. No. 60/853,968, first author RossTye, filed on Oct. 23, 2006, all of which applications are incorporatedherein by reference.

INTRODUCTION

This invention relates to nitric oxide-blocked cross linked tetramerichemoglobins, more specifically to a carboxamidomethylated cross linkedtetrameric hemoglobin, which has low reactivity with nitric oxide (NO),and which is cross linked to stabilize the tetramer for in-vivoapplications. Methods of preparation and use as blood volume expansionagents and as oxygen delivery therapy agents are also disclosed.

BACKGROUND OF THE INVENTION

One of the limitations on the use of blood in an emergency setting is arequirement to type and cross-match the blood to minimize the risk oftransfusion reactions. Type and cross-matching may require at least 10minutes and a complete type and cross-match can take up to an hour.Furthermore, the risk of HIV transmission has been estimated to be 1 in500,000 units of blood and the risk of hepatitis C transmission has beenestimated to be 1 in 3,000 units. The safety of blood supply and bloodlogistics are critical issues in developing countries, where the risk ofinfectious disease transmission as well as the risk of outdated supplyis relatively higher. Up to 25% of the blood is discarded in developingcountries because of the presence of infectious disease. Hence, thereare pressing factors to find blood substitutes or artificial bloodcompositions that avoid disease transmission and provide rapid responseto improve chances of survival.

Two aspects of artificial blood use in clinical settings are volumeexpansion and oxygen therapeutics. Volume expander agents are inert,merely increasing, blood volume, and thus allow the heart to pump fluidefficiently. Oxygen therapeutics mimic human blood's oxygen transportability. Oxygen therapeutics can be divided in two categories based ontransport mechanism: perfluorocarbon based, which function by simpledissolution of oxygen, and hemoglobin protein based, which transportsoxygen by facilitated capture and release. In hemoglobin-based products,pure hemoglobin (Hb) separated from red blood cells (RBCs) may not beuseful for a number of reasons, including instability, induction ofrenal toxicity, and unsuitable oxygen transport and deliverycharacteristics when separated from red blood cells.

Hemoglobin based oxygen therapeutics have been shown to exert variousdegrees of vasoactive effects both in animal and human studies (Winslowet al., Adv Drug Del Rev 2000; 40: 131-42; Stowell et al., Transfusion2001; 41: 287-99; Spahn et al., News Physiol Sci 2001; 16: 38-41; Spahnet al., Anesth Analg 1994; 78: 1000-21; Kasper et al., Anesth Analg1996; 83: 921-7; Kasper et al., Anesth Analg 1998; 87: 284-91; Levy etal., J Thorac Cardiovasc Surg 2002; 124: 35-42;). This vasoactivity maybe due to the effects of these products in binding intracellular NO(Kasper et al., Anesth Analg 1996; 83: 921-7; Dietz et al., Anesth Analg1997; 85: 265-273; Schechter et al., N Engl J Med 2003; 348: 1483-5),endothelial release (Gulati et al., Crit. Care Med 1996; 24: 137-47), orsensitization of peripheral α-adrenergic receptors (Gulati et al., J LabClin Med 1994; 124: 125-33). Alternatively, the increasedvasoconstrictive effects could be due to increases in the rate of oxygenrelease, secondary to the administration of these products, at a higherconcentration than RBCs, resulting in vasoconstriction (Winslow et al.,J Intern Med 2003; 253: 508-17; McCarthy et al., Biophys Chem 2001; 92:103-17; Intaglietta et al., Cardiovasc Res 1996; 32: 632-43; Vandegriffet al., Transfusion 2003; 43: 509-16).

The ability of stroma-free Hb solutions to induce blood pressureincreases has been known. It has been demonstrated that somecross-linked Hb solutions could increase mean arterial pressure as muchas 25-30% in a dose-dependent manner within 15 min of administration andthat the effect could last as long as 5 h.

Vasoconstriction may be due to NO scavenging by the hemoglobin basedtherapeutic (Katsuyama et al., Artif Cells Blood Substit ImmobilBiotechnol 1994; 22:1-7; Schultz et al., J Lab Clin Med 1993;122:301-308, hereby incorporated by reference in its entirety).Vasoconstriction could be also caused by the contamination of thehemoglobin by phospholipids and endotoxin. Although the remainingphospholipids and endotoxin contamination during Hb purification maycause hemodynamic effects (Macdonald et al., Biomater Artif Cells ArtifOrgans 1990; 18: 263-282), it is less likely that this contamination bethe major factor explaining the potent vasoactive effect of some ofthese products (Gulati et al., Life Sci 1995; 56: 1433-1442).

NO is a smooth-muscle relaxant that functions via activation ofguanylate cyclase and the production of cGMP or by direct activation ofcalcium-dependent potassium channels. The increase in the free Hb canresult in an increase in the NO binding. The increase in the NO bindingcan result in transient and in repeat dosing, sustained hemodynamicchanges responding to vasoactive substances or the lack of vasoactiveregulatory substances. In some circumstances the lack of nitric oxidemay lead to blood pressure increases and if prolonged, hypertension. Ithas been demonstrated that NO may bind to the reactive sulfhydryls of Hband may be transported to and from the tissues in a manlier analogous tothe transport of oxygen by heme groups (Jia et al., Nature 1996;80:221-226).

Nitric oxide along with precapillary sphincter movement are regulatorsof the arteriolar perfusion of any tissue. Nitric oxide is synthesizedand released by the endothelium in the arterial wall, where it can bebound by hemoglobin in red blood cells. When a tissue is receiving highlevels of oxygen, nitric oxide is not released and the arterial wallmuscle contracts making the vessel diameter smaller, thus decreasingperfusion rate and cause a change in cardiac output. When demand foroxygen increases, the endothelium releases nitric oxide, which causesvasodilatation. The nitric oxide control of arterial perfusion operatesover the distance that NO diffuses after release from the endothelium.Nitric oxide is also needed to mediate certain inflammatory responses.For example, nitric oxide produced by the endothelium inhibits plateletaggregation. Consequently, as nitric oxide is bound by cell-freehemoglobin, platelet aggregation may be increased. As plateletsaggregate, they release potent vasoconstrictor compounds such asthromboxane A₂ and serotonin. These compounds may act synergisticallywith the reduced nitric oxide levels caused by hemoglobin scavengingresulting in significant vasoconstriction. In addition to inhibitingplatelet aggregation, nitric oxide also inhibits neutrophil attachmentto cell walls, which in turn may lead to cell wall damage. Becausenitric oxide binds to hemoglobin inside the red blood cell, it isexpected that nitric oxide may bind to free Hb (stroma free crosslinkedtetrameric Hb) as well.

In many formulations free Hb and stabilized hemoglobin infusions appearto be linked to vasoconstriction of the blood vessels, resulting inextremely high blood pressures. The hemoglobin moiety of these productscan diffuse into the endothelial lining of the vascular wall and act asa sink in binding and removing NO which is needed for maintaining thenormal tone of the vascular wall. This can result in vasoconstriction ofthe smooth muscle cells of the vascular wall. The free Hb solution canleak into the surrounding tissues. Also, the extent of vasoconstrictionwhich occurs subsequent to administration of different molecular sizehemoglobin-based therapeutic bears an inverse relationship to themolecular size of the product used, i.e. infusion of larger oxygencarriers results in less vasoconstriction and hypertension (Sakai, etal. Am J Physiol 2000; 279: H908-15). The smaller sized Hb molecule maybe the most permeable and may show a higher level of vasoconstrictionand hypertension (Faivre-Fiorina et al., Am J Physiol Heart Circ Physiol1999; 276: H766-70). In rabbit models, transfusion of free Hb throughthe ear vein has caused cerebral vasculature ischemia and death.Therefore, it is important to minimize the impact of administration ofmost free Hb on the arterial system during administration. Vasoactiveagents such as verapamil, atenocard, sildenafil citrate, etc., may beadministered to the patient prior to free Hb infusion. This is intendedto ensure that the arterial system is minimally changed during infusion.Nitric oxide and verapamil are preferred vasoactive agents. Slow channelcalcium blockers (or a selective inhibitor of cyclic guanosinemonophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), such assildenafil citrate) may also be helpful in the prevention of the severevasoconstriction. However, a slower infusion rate may not be possiblewith respect to a trauma patient when demand for volume is acute andcritical.

One mechanism of modifying the NO scavenging properties of hemoglobinbased therapeutics is blocking of NO binding sites on these molecules.Unprotected thiol on the cysteine moiety of the hemoglobin may bind withNO. Protection of thiol or sulfhydryl groups in the hemoglobin moleculemay prevent the binding of NO to the hemoglobin at the thiol site andhence prevent an acute vasoactive response of the blood vessels causinga hypertensive reaction. The prevention of NO binding to hemoglobinbased therapeutics may also prevent interference with normal plateletaggregation and neutrophil migration when this class of therapeutics isadministered.

Therefore, some of the desirable characteristics of hemoglobin basedoxygen delivery therapeutics are: toxicity-free, lack of induction ofharmful immunogenic response, satisfactory oxygen carrying and deliverycapacity, suitable circulatory persistence to permit effectiveoxygenation of tissues, long shelf life, capacity for storage at roomtemperature, absence of viral or other pathogens to prevent diseasetransmission, elimination of the requirement for blood typing, andcapacity for deployment by first responders such as, paramedics, frontline military medics etc. These characteristics provide a rapid, saferesponse to blood loss and the immediate support of tissue metabolicneeds, thus improving the chances for survival.

The present invention disclosed herein provides compositions,characteristics and methods to prepare deoxygenated, endotoxin free,stroma free, thiol blocked, cross-linked tetrameric hemoglobin which haslow reactivity with Nitric Oxide (NO), and the tetrameric structures isstabilized by cross-linking. In particular a carboxamidomethylated crosslinked tetrameric hemoglobin is provided as a stable NO blockedtetrameric Hb of the invention, as well as methods for its production. Aprocess and methods of preparation of stable NO-blocked tetrameric Hb ofthe invention are disclosed as well as methods of use as blood volumeexpansion agents and as oxygen delivery therapy agents.

SUMMARY OF THE INVENTION

The present invention relates to a proteinaceous iron containingcompound having a molecular weight distribution in the range of about60,000 daltons to about 500,000 daltons and having at least one cysteinemoiety wherein the cysteine moiety includes a thiol protecting groupsuch that the proteinaceous compound has a reduced ability to bindnitric oxide at the cysteine site(s). In some embodiments, theproteinaceous iron containing compound transports oxygen with a p50 ofabout 20 mm Hg to about 45 mmHg. In some embodiments, the proteinaceousiron containing compound is incapable of binding nitric oxide at thecysteine site(s).

Another aspect of the invention relates to a composition comprising aproteinaceous iron containing compound having a molecular weightdistribution in the range of about 60,000 daltons to about 500,000daltons and having at least one cysteine moiety wherein the cysteinemoiety includes a thiol protecting group such that the proteinaceouscompound has a reduced ability to bind nitric oxide at the cysteinesite(s).

Yet another aspect of the invention relates to a composition comprisinga proteinaceous iron containing compound having a molecular weightdistribution in the range of about 60,000 daltons to about 500,000daltons and having at least one cysteine moiety wherein the cysteinemoiety includes a thiol protecting group such that the proteinaceouscompound has a reduced ability to bind nitric oxide at the cysteinesite(s) and wherein said compound is a cross-linked tetramerichemoglobin.

Another aspect of the invention relates to a process for a preparationof a tetrameric hemoglobin wherein the hemoglobin has a thiol protectinggroup attached to a cysteine group of the hemoglobin comprising: (a)removing endotoxin and other lipopolysaccharides from a preparationcontaining red blood cells; (b) lysing the red blood cells; (c)separating hemoglobin by removing stroma from the lysed red blood cells;(d) optionally removing oxygen from the hemoglobin; (e) adding to ahemoglobin solution a reagent which provides a thiol protecting groupfor a cysteine of the hemoglobin, and (f) separating a hemoglobin whichhas a thiol protecting group attached to a cysteine.

In some embodiments of the aforementioned aspect of the invention, theprocess further comprises: (a) optionally removing oxygen from thehemoglobin which has a thiol protecting group attached to a cysteine;and crosslinking the hemoglobin which has a thiol protecting groupattached to a cysteine of the hemoglobin, yielding a stable NO-blockedtetrameric Hb of the invention, which is cross-linked.

In another aspect of the invention, a method for producing tetramerichemoglobin is provided wherein a thiol protecting group is attached to acysteine in the hemoglobin, by a process comprising: (a) removingendotoxin from a preparation containing red blood cells; (b) lysing saidred blood cells; (c) separating hemoglobin by removing stroma from saidlysed red blood cells; (d) optionally deoxygenating said hemoglobin; (e)adding to a hemoglobin solution a reagent which provides a thiolprotecting group for a cysteine of said hemoglobin, and (f) separating ahemoglobin which has a thiol protecting group attached to a cysteine ofthe hemoglobin.

In another aspect of the invention, a method for producing a crosslinked tetrameric hemoglobin is provided, by a process comprising:optionally removing oxygen from the product of the method for productingtetrameric hemoglobin; and crosslinking said product.

Yet another aspect of the invention relates to a method for producing aNO-blocked tetrameric hemoglobin wherein a thiol protecting group isattached to a cysteine in the hemoglobin, by a process comprising: (a)adding to the hemoglobin solution a reagent which provides a thiolprotecting group for a cysteine of the hemoglobin, and (b) separating ahemoglobin which has a thiol protecting group attached to a cysteine ofthe hemoglobin. In some embodiments the hemoglobin is furthercross-linked.

Another aspect of the invention relates to a method of supplementing theblood volume of a mammal comprising administering to the mammal acomposition comprising a proteinaceous iron containing compound having amolecular weight of about 60,000 daltons to about 500,000 daltons andhaving at least one cysteine moiety wherein the cysteine moiety includesa thiol protecting group such that the proteinaceous compound hasreduced ability to bind nitric oxide at the cysteine site(s), andfurther comprises a pharmaceutically acceptable carrier. In someembodiments the proteinaceous iron containing compound is cross-linked.

In another aspect of the invention, a method of treating a mammalsuffering from a disorder is provided, comprising administering acomposition comprising a proteinaceous iron containing compound having amolecular weight of about 60,000 daltons to about 500,000 daltons andhaving at least one cysteine moiety where the cysteine moiety includes athiol protecting group such that the proteinaceous compound has reducedability to bind nitric oxide at the cysteine site(s). In someembodiments the proteinaceous iron containing compound is cross-linked.

In another aspect of the invention, a method is provided for perfusingan organ comprising administering an effective amount of the stableNO-blocked tetrameric hemoglobins of the invention, which can further beperformed in-vivo or ex-vivo.

In some embodiments, the proteinaceous iron containing compoundincreases oxygen offloading capacity relative to native, cell freehemoglobins. In some embodiments, the proteinaceous iron containingcompound increases oxygen delivery ability. In some embodiments, thecrosslinked tetrameric hemoglobin is materially reduced in its abilityto bind nitric oxide. In some embodiments the cross linked tetramerichemoglobin is incapable of binding nitric oxice. In some preferredembodiments, the crosslinked tetrameric hemoglobin transports oxygenwith a p50 of about 20 mm Hg to about 45 mm of Hg. In some embodimentsthe proteinaceous iron containing compound transports oxygen with a p50of about 20 mm Hg to about 45 mm of Hg.

In some embodiments of the invention, the proteinaceous iron containingcompound is a thiol-protected hemoglobin. In some embodiments, theproteinaceous iron containing compound is a cross-linked tetramerichemoglobin. In some embodiments, the proteinaceous iron containingcompound has been crosslinked with his 3′,5′ dibromo salicyl fumarate.In some embodiments, the hemoglobin has been modified by reaction withpyridoxal-5′-phosphate. In some embodiments, the hemoglobin ismammalian. In some embodiments, the hemoglobin is human hemoglobin. Insome embodiments, the hemoglobin is bovine (i.e. bovine (genus bos) orbison (genus bison)) or porcine hemoglobin. In some preferredembodiments, the hemoglobin is non-pyrogenic, endotoxin free, oxygenfree and stroma free, enzyme free, and with low induction of negativeimmunogenic reactions.

In some preferred embodiments, oxygen is removed from hemoglobin whichmay or may not have a thiol protecting group attached to a cysteine ofthe hemoglobin. In some embodiments the oxygen is removed by contactormembrane technology.

In another aspect of the invention, the proteinaceous iron containingcompound of the invention is a thiol blocked stroma free hemoglobin thatmay be safely stored for extended periods. This thiol blocked stromafree hemoglobin may be a stable intermediate which can endure packaging,shipping and further handling to yield another hemoglobin composition ofthe invention. In some embodiments the stable intermediate is furtheroptionally deoxygenated, cross-linked, and purified to remove excessreagents and byproducts of the reaction, for example, dibromo salicylicacid. In some embodiments the stable NO blocked tetrameric hemoglobin ispackaged.

In other embodiments of the invention, the compound is non-pyrogenic,endotoxin free, and stroma free. In some embodiments of the invention isproteinaceous compound is of low viscosity. In some embodiments theproteinaceous compound of the invention is oxygen free.

In some embodiments, the reagent that provides a thiol protecting groupis selected from the group consisting of 4-pyridylmethyl chloride,alkoxyalkylchloride, dimethoxymethane, N-(hydroxymethyl)acetamide,triphenylmethyl chloride, acetyl chloride, acetic anhydride,haloacetamide, iodoacetate, benzyl chloride, benzoyl chloride,di-tert-butyl dicarbonate, p-hydroxyphenacyl bromide, p-acetoxybenzylchloride, p-methoxybenzyl chloride, 2,4-dinitrophenyl fluoride,tetrahydropyran, acetamidohydroxymethane, acetone,bis-carboethoxyethene, 2,2,2-trichloroethoxycarbonyl chloride,tert-butoxycarbonyl chloride, alkyl isocyanate, and alkoxyalkylisocyanate. In some preferred embodiments, the haloacetamide isiodoacetamide. In some embodiments, the thiol protecting group isselected from the group consisting of 4-pyridylmethyl,acetylaminomethyl, alkoxyalkyl, triphenylmethyl, derivatives ofcarboxymethyl, carboxamidomethyl, acetyl, benzyl, benzoyl,tert-butoxycarbonyl, p-hydroxyphenacyl, p-acetoxybenzyl,p-methoxybenzyl, 2,4-dinitrophenyl, isobutoxymethyl, tetrahydropyranyl,acetamidomethyl, benzamidomethyl, bis-carboethoxyethyl,2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, N-alkyl carbamate,and N-alkoxyalkyl carbamate. In some embodiments, the thiol protectinggroup is a carboxamidomethyl group.

Some embodiments of the invention provide compositions comprising theproteinaceous iron containing compound and a pharmaceutically acceptablecarrier. In some embodiments provide a container containing acomposition comprising the proteinaceous compound of the invention,optionally comprising a pharmaceutically acceptable carrier.

In some embodiments, the mammal suffers from acute anemia, anemiarelated conditions, hypoxia of ischemia. In some embodiments, the mammalneeds volume transfusion of a blood substitute for transport of oxygen.In some embodiments, the mammal is in trauma and has suffered an acutevolume loss.

In some embodiments of the methods of the invention, administration ismade by implant, injection or transfusion.

In other embodiments of the method of the invention, the mammals aresuffering from a disorder including anemia, anemia related conditions,hypoxia and ischemia. The anemia and anemia related conditions may becaused by renal failure, diabetes, AIDS, chemotherapy, radiationtherapy, hepatitis, G.I. blood loss, iron deficiency, or menorrhagia. Insome embodiments of the invention, the method includes administeringerythropoietin therapy

In some embodiments of the method of the invention, the disorder beingtreated is ischemia, which is caused by burns, stroke, emerging stroke,transient ischemic attacks, myocardial stunning and hibernation, acuteangina, unstable angina, emerging angina, or infarct. In otherembodiments of the method, the disorder is carbon monoxide poisoning.

In other embodiments of the method of the invention, the disorder thatthe mammal is treated for is recovery after surgery. In some otherembodiments of the method of the invention, the disorder is diabeticwound healing. In yet other embodiments of the method the disorder issickle cell anemia, and the administration may further be made prior tosurgery. In other embodiments of the method of the invention, thedisorder is acute coronary syndrome. In other embodiments of the methodof the invention, the disorder is cardiogenic shock.

In some embodiments of the method of the invention the proteinaceousiron containing compound is administered to a mammal in need of a bloodtransfusion. In some embodiments of the invention, the mammal issuffering from trauma. In some embodiments of the method, the disorderthat the mammal is suffering from is lack of oxygen delivery capacity iscaused by environmental stress or physical stress.

In other embodiments of the method of the invention, the proteinaceousiron containing compound is administered in combination with radiationtherapy. In yet other embodiments of the invention, the method furthercomprises administering to said mammal an oxygen dependentpharmaceutical agent.

In some embodiments of the method of the invention, administering theproteinaceous iron containing compound to said mammal permitsvisualization of intravascular space in-vivo, while maintainingoxygenation of the tissue within the viewing field.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a flow chart showing the steps of the methods as disclosedherein.

FIG. 2 depicts the time course of a lysis experiment showing resistanceof WBC lysis.

FIG. 3 depicts the size standards used in electrophoretic separations asdisclosed within.

FIG. 4A depicts overlays of electrophoretic separations of nativehemoglobin, dXCMSFH, and size standard in the range around 20 KDa.

FIG. 4B depicts overlays of the electrophoretic separation of nativehemoglobin, dXCMSFH, and size standards for the full electropherogram.

FIG. 5 depicts the HLPC size exclusion separation of products of thecross linking reaction.

FIG. 6 shows oxygen affinity curves for bovine whole blood, stroma freeHb, cross linked hemoglobin, and fresh human blood.

FIGS. 7A-D depict the cardiac output, systemic vascular resistance, andmean arterial pressure, respectively, in a pig safety trial.

DETAILED DESCRIPTION OF THE INVENTION

The term “endotoxin free” or its grammatical equivalents as used herein,means a hemoglobin that has been treated to reduce exposure to and toremove substantially or completely all endotoxin as measured by a verysensitive assay technique such as a tubidometric assay or a chromogenicassay. These methods are capable of detecting less than 0.05 EU per ml.Thus, endotoxin-free hemoglobin can have less than or the equivalent ofthe amount of endotoxin present in Water for Injection (WFI).

The term “non-pyrogenic” or its grammatical equivalents as used herein,means a hemoglobin that may be administered to a mammal without causingIL-8 overproduction, complement activation, platelet activation,inflammatory response or a febrile reaction.

The term “oxygen free”, “deoxygenated”, or its grammatical equivalentsas used herein, means a hemoglobin that has been treated to removesubstantially or completely all oxygen bound to the heme pocket.Oxygen-free hemoglobin thus is substantially or completely in the higherenergy “tense” or “T” configuration.

The term “stroma free” or its grammatical equivalents as used herein,means a hemoglobin that has been treated or processed to removesubstantially or completely all stromal material, such that thepreparation no longer exhibits the immunoreactivity to red cell surfacetype antigens characteristic of RBC membranes. Stroma are the cellmembrane structural proteins and removing stroma also will removeantigens associated with the cell membrane. Stroma-free hemoglobintherefore substantially or completely lacks the toxic and/or pyrogenicproperties associated with preparations of hemolyzed red blood cellsstill containing portions of the lipid membrane surrounding thehemoglobin protein, and thus after molecular stabilization, thisstabilized stroma free hemoglobin can be administered to an individualwithout causing transfusion reaction toxicity or inflammatory reaction.

The term “NO-blocked tetrameric Hb” refers to endotoxin-free,stroma-free thiol blocked tetrameric hemoglobins of the presentinvention. The term “stable NO-blocked tetrameric Hb” refers to thecross-linked, endotoxin-free, stroma free, thiol blocked hemoglobins ofthe present invention.

The term “dNO-blocked tetrameric Hb” refers to the deoxygenatedendotoxin-free, stroma-free, thiol blocked, cross-linked hemoglobins ofthe present invention. One embodiment of this class of compounds is“dXCMSFH”, which is a specific example of a deoxygenated endotoxin-free,stroma-free, carboxamidomethylated cross linked hemoglobin of thepresent invention.

The term “dTBSFH” refers to the deoxygenated endotoxin-free,stroma-free, thiol-blocked uncross-linked hemoglobin of the presentinvention.

The term “dCMSFH refers to the deoxygenated endotoxin-free, stroma free,carboxamidomethylated uncross-linked hemoglobin of the invention and isa specific example of a dTBSFH.

The term “mammal” refers to both human and non-human animals.

The compositions and methods of the present invention relate to athiol-protected, cross-linked tetrameric hemoglobin (stable NO blockedHb) where at least one cysteine moiety in the hemoglobin moleculeincludes a thiol protecting group, for example, a carboxamidomethylgroup, such that the thiol group in the cysteine moiety is not availablefor binding with nitric oxide (NO). Preferably, at least two cysteinemoieties in the hemoglobin are protected with a thiol (or sulfhydryl)protecting group such that the thiol group in the cysteine moiety is notavailable for binding with nitric oxide (NO). These NO-blockedhemoglobins disclosed herein prevent vasoactive reactions of bloodvessels when administered. The stable NO-blocked tetrameric hemoglobinsof the invention are further cross linked to provide an oxygen carryingcapacity with a p50 of about 20 mm Hg to about 45 mm Hg and an extendedcirculatory half life. Preferably, the hemoglobin is non-pyrogenic,endotoxin free, oxygen free, and stroma free. Therefore, thiol-protectedcross-linked hemoglobins of the present invention (a stable NO-blockedtetrameric Hb) have high oxygen exchange capacity and are functionallysuperior to native hemoglobin.

I. HEMOGLOBIN COMPOSITIONS 1. Hemoglobin Sources and Molecular Structure

Hemoglobin (or blood or RBCs which it may be isolated from) used in thepresent-invention may be obtained from a variety of mammalian sources,such as, for example, human, bovine (genus bos), bison (genus bison),ovine (genus ovis), porcine (genus sus) sources, other vertebrates ortransgenically-produced hemoglobin. Alternatively, the stroma-freehemoglobin used in the present invention may be synthetically producedby a bacterial, or more preferably, by a yeast, mammalian cell, orinsect cell expression vector system (Hoffman, S. J. et al., U.S. Pat.No. 5,028,588 and Hoffman, et al., WO 90/13645, both herein incorporatedby reference). Alternatively, hemoglobin can be obtained from transgenicanimals; such animals can be engineered to express non-endogenoushemoglobin (Logan, J. S. et al. PCT Application No. PCT/US92/05000;Townes, T. M. et al., PCT Application No. PCT/US/09624, both hereinincorporated by reference in their entirety). Preferably, thestroma-free hemoglobin used in the present invention is isolated frombison, bovine or human sources.

The genus bos includes, subgenus bos including bos taurus (westerncattle, including oxen and aurochs) and bos aegyptiacus; subgenus bibosincluding bos frontalis (gaur, gayal or Indian bison) and bos javanicus(banteng); subgenus novibos including bos sauveli (kouprey or grey ox),and; subgenus poephagus including bos grunniens (yak; also bos mutus).The bos taurus, includes similar types from Africa and Asia such as, bosindicus, the zebu; and the bos primigenius, the aurochs. The bos gurusincludes subspecies, bos gaurus laosiensis, bos gaurus gaurus (such asin India, Nepal) also called “Indian bison”, bos gaurus readei, bosgaurus hubbacki (such as in Thailand, Malaysia), and bos gaurusfrontalis, a domestic gaur, or a gaur-cattle hybrid breed.

Bison is a taxonomic genus containing six species within the subfamilybovinae. The bison may be called buffalo in Asia (such as water buffalo)and Africa (such as African buffalo). The genus bison includes speciessuch as, bison latifrons (long-horned bison), bison antiquus, bisonoccidentalis, bison priscus, bison bison, bison bison bison, bison bisonathabascae, bison bonasus, bison bonasus bonasus, bison bonasuscaucasicus, and bison bonasus hungarorum. In some embodiments of thepresent invention, the hemoglobin is from genus bos or bison It shall beunderstood that any mammalian species may be used as a source ofhemoglobin and is within the scope of the present invention.

Bovine Hb is easier to obtain and more abundant than human Hb. Typicallyhuman Hb extracted from outdated RBCs is used for Hb-based artificialblood research. However, outdated RBCs are not available in sufficientquantities to produce large amounts of viable oxygen deliverytherapeutics or blood substitutes.

Hemoglobin, whether derived from an animal, synthetic or recombinant,may be composed of the “naturally existing” hemoglobin protein, or maycontain some or be entirely composed of, a mutant hemoglobin protein.Preferred mutant hemoglobin proteins include those whose mutationsresult in more desirable oxygen binding/release characteristics.Examples of such mutant hemoglobin proteins include those provided byHoffman, S. L. et al. (U.S. Pat. Nos. 5,028,588 and 5,776,890) andAnderson, D. C. et al. (U.S. Pat. Nos. 5,844,090 and 5,599,907), allherein incorporated by reference in their entirety.

Hemoglobin or haemoglobin (Hb) is a proteinaceous heme iron-containingcompound having a molecular weight of about 60,000 daltons whichtransports oxygen in the red blood cells of the blood in mammals andother animals. Hemoglobin transports oxygen from the lungs to the restof the body, such as to the muscles, wherein it releases part of theoxygen load. The hemoglobin molecule is an assembly of four globularprotein subunits. Each subunit is composed of a protein chain tightlyassociated with a non-protein heme group. Each individual protein chainarranges in a set of α-helix structural segments connected together in a“myoglobin fold” arrangement, so called because this arrangement is thesame folding motif used in the heme/globin proteins. This foldingpattern contains a pocket which is suitable to strongly bind the hemegroup. A heme group consists of an iron atom held in a heterocyclicring, known as a porphyrin. These iron atoms are the sites of oxygenbinding. The iron atom is bonded equally to all four nitrogens in thecenter of the ring, which lie in one plane. Two additional bondsperpendicular to the plane on each side can be formed with the iron toform the fifth and sixth positions, one connected strongly to theprotein, the other available for binding of oxygen. The iron atom caneither be in the Fe²⁺ or Fe³⁺ state, but ferrihaemoglobin(methemoglobin) (Fe³⁺) cannot bind oxygen.

In adult humans, the predominate hemoglobin type is a tetramer (whichcontains 4 subunit proteins) called hemoglobin A, consisting of two αand two β subunits non-covalently bound, each made of 141 and 146 aminoacid residues, respectively. This is denoted as α₂β₂. The subunits arestructurally similar and about the same size. Each subunit has amolecular weight of about 16,000 daltons, for a total molecular weightof the tetramer of about 64,000 daltons. The four polypeptide chains arebound to each other by salt bridges, hydrogen bonds and hydrophobicinteractions. There are two kinds of contacts between the α and the βchains: α₁β₁ and α₁β₂. However, adult hemoglobin may also comprise δglobin subunits. The δ globin subunit replaces β globin and pairs with αglobin as α₂δ₂ to form hemoglobin A2.

Bovine Hb is structurally similar to human Hb. The bovine Hb alsocontains two α chains and two β chains, with similar molecular weightdistribution.

2. Protecting the Sulfhydryl Groups of the Cysteine of the Hemoglobin

A thiol group of a cysteine moiety in a hemoglobin may bind to nitricoxide and may result in transient or sustained changes in hemodynamicproperties of blood or vasoactive substances and may lead tohypertensive reactions. This occurrence can be avoided by protecting thethiol group of the cysteine moiety in the hemoglobin such that theresulting hemoglobin is incapable of binding with NO.

Bovine hemoglobin contains only two thiol groups (Cys 93 on each of thebeta chains) which are involved in binding NO. Hence preferably, boththiol groups are protected with a thiol protecting group in bovine Hb.Human hemoglobin contains six thiol groups (α Cys 104, β Cys 93, and βCys 112), and at least two of which (Cys 93 on each of the beta chains)are involved in binding NO. Preferably these two thiols are protectedand up to six thiol groups may be protected with a thiol protectinggroup in human Hb.

The SFH of the present invention can be reacted with various reagents toresult in protection of the thiol group in the cysteine moiety of thehemoglobin. Without limiting the scope of the present invention, all thereagents known in the art for the protection of a functional group suchas, but not limited to, hydroxyl, thiol, or carboxyl, are included inthe present invention.

Some of the examples of the reagents include, but are not limited to,4-pyridylmethyl chloride, alkoxyalkylchloride, dimethoxymethane,N-(hydroxymethyl)acetamide, triphenylmethyl chloride, acetyl chloride,2-chloroacetic acid, acetic anhydride, haloacetamide such as,iodoacetamide, bromoacetamide, chloroacetamide, or fluoroacetamide,haloacetate such as iodoacetate, bromoacetate, chloroacetate, orfluoroacetate, benzyl chloride, benzoyl chloride, di-tert-butyldicarbonate, p-hydroxyphenacyl bromide, p-acetoxybenzyl chloride,p-methoxybenzyl chloride, 2,4-dinitrophenyl fluoride, tetrahydropyran,acetamidohydroxymethane, acetone, bis-carboethoxyethene,2,2,2-trichloroethoxycarbonyl chloride, tert-butoxycarbonyl chloride,alkyl isocyanate, and alkoxyalkyl isocyanate. In a preferred embodiment,the reagent is haloacetamide. In a further preferred embodiment, thereagent is iodoacetamide. It is understood that any reagent known in theart that can be used for carboxamidomethylation of the thiol group inthe cysteine moiety of the hemoglobin is within the scope of the presentinvention.

Without limiting the scope of the present invention, all the protectinggroups known in the art for the protection of a functional group suchas, but not limited to, hydroxyl, thiol, or carboxyl, are included inthe present invention. Some of the examples of the protecting groupinclude, but are not limited to, 4-pyridylmethyl, acetylaminomethyl,alkoxyalkyl, triphenylmethyl, carboxamidomethyl, acetyl, benzyl,benzoyl, tert-butoxycarbonyl, p-hydroxyphenacyl, p-acetoxybenzyl,p-methoxybenzyl, 2,4-dinitrophenyl, isobutoxymethyl, tetrahydropyranyl,acetamidomethyl, benzamidomethyl, bis-carboethoxyethyl,2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, N-alkyl carbamate,and N-alkoxyalkyl carbamate. In a preferred embodiment, the protectinggroup is carboxamidomethyl such that the protection of the thiol groupin the cysteine moiety of the hemoglobin results in a non-pyrogenic,endotoxin free, stroma free, carboxamidomethylated Hb (CMSFH or a stableNO blocked Hb). More generally, protection of the thiol group incysteine(s) of hemoglobin results in a non-pyrogenic, endotoxin free,stroma free thiol blocked Hb (TBSFH or an NO blocked Hb).

3. Oxygen Affinity Modulation and Stabilization by Cross Linking withinTetrameric Hemoglobin.

Bovine Hb and human Hb differ in the way in which oxygen affinity ismodulated. In the tetrameric form of normal adult human hemoglobin, thebinding of oxygen is a cooperative process. The binding affinity ofhemoglobin for oxygen is increased by the oxygen saturation of themolecule. As a consequence, the oxygen binding curve of hemoglobin issigmoidal, or S-shaped. This positive cooperative binding may beachieved through steric conformational changes of the hemoglobin proteincomplex. When one subunit protein in hemoglobin becomes oxygenated, itinduces a conformational or structural change in the whole complexcausing the other subunits to gain an increased affinity for oxygen.

When hemoglobin binds oxygen, it shifts from the high energy “tense” or“T” state (deoxygenated or oxygen free) to the lower energy “relaxed” or“R” state (oxygenated). Human α and β globin genes have been cloned andsequenced (Liebhaber et al., Proc. Natl. Acad. Sci. (U.S.A). 77:7054-58(1980); Marotta et al., J. Biol. Chem. 252:5040-43 (1977); and Lawn etal., Cell 21:647 (1980), all of which are incorporated by reference intheir entirety). The tetrameric structure of human T statedeoxyhemoglobin has increased stability from six ionic bonds and whilein the T state, hemoglobin is effectively prevented from disassociatinginto dimers. In this conformation, the beta cleft contact area betweenthe two beta chains (also known as the beta pocket, phosphate pocket,and 2,3-diphosphoglycerate binding site) in deoxyhemoglobin issubstantially different than in oxyhemoglobin. The changed conformationof the beta cleft in the T state is believed to explain the decreasedoxygen affinity stabilized by 2,3-diphosphoglycerate. The T state ofhemoglobin is stable and resistant to denaturation.

Inside red blood cells, the binding of 2,3-diphosphoglycerate to itsbinding site within human hemoglobin decreases the hemoglobin's oxygenaffinity to a level compatible with oxygen transport and delivery in aphysiologic range of pH 7.2 to 7.4. The binding of2,3-diphosphoglycerate to hemoglobin is weak and may require highconcentrations (i.e., concentrations approaching 1M or more) in order tomodify the oxygen affinity of hemoglobin. For example, in people acutelyacclimated to high altitudes, the concentration of2,3-diphosphoglycerate (2,3-DPG) in the blood is increased, which allowsthese individuals to release a larger amount of oxygen to tissues underconditions of lower oxygen tension.

Thus, when the red blood cells are ruptured to produce stroma freehemoglobin (SFH), the 2,3-diphosphoglycerate may not be retained inclose proximity to the hemoglobin and may disassociate from thehemoglobin. As a consequence, unless further modified, Human SFH mayexhibit a higher affinity for oxygen than does hemoglobin in RBCs. Thep50 of stroma free human hemoglobin in solution can be approximately 12to 17 mm Hg as compared to native, RBC associated hemoglobin p50 ofapproximately 27 mm Hg. The increased affinity of the SFH for oxygen,under physiological conditions, may prevent high capacity release of thebound oxygen to the tissues.

In contrast, bovine Hb, possessing a further internal salt bridge, hasits affinity for oxygen affected by the ionic strength of the localenvironment. Bovine hemoglobin does not require 2,3-DPG to maintain ap50 for oxygen in the range of 30 mm Hg to 40 mm Hg. An advantage toaffinity modulation by altering ionic strength versus that induced by2,3-DPG binding is that sufficient concentration of ionic species isgenerally present in plasma while 2,3-DPG is only contained within RBCs.Thus, the oxygen affinity of acellular bovine Hb can be modulated moreeasily than acellular human Hb.

This advantage of modulation of affinity by general ionic interactioncan be built back into human Hb by reacting it withpyridoxal-5-phosphate (PLP). PLP modifies human Hb by introducing anegative charge near a penultimate β chain histidine residue and byremoving a positive charge at the amino terminal end of the same chain,An altered human Hb of this class can now respond more similarly tobovine Hb to charged species in the local environment and not solelydepend on binding of 2,3-DPG to affect oxygen affinity.

Within the RBC, the association of the α chain with its corresponding βchain is very strong and does not disassociate under physiologicalconditions. The association of one α/β dimer with another α/β dimer,however, is fairly weak and outside of the RBC, the two dimers maydisassociate even under physiological conditions. Upon disassociation,the dimer is filtered through the glomerulus. The rapid clearing ofstroma free hemoglobin (SFH) by the kidney is a consequence of itsquaternary molecular arrangement.

To avoid such removal of human and bovine hemoglobin alike, cell-freehemoglobin can be conjugated or cross-linked by various methods known inthe art. One of the methods is by conjugating Hb to another moleculesuch as polyethylene glycol (PEG), which forms a hydrophilic shieldaround the Hb molecule and simultaneously increases its size which inturn increases its circulatory half-life. Hb can also be cross-linkedintramolecularly to prevent dissociation of the tetramer into αβ dimersand/or cross-linked intermolecularly to form polymers which alsoincreases the oxygen carrier's size and thus increases its circulatoryhalf-life. Using site-specific cross-linking reagents, intramolecularcovalent bonds may be formed, which may convert Hb into a stabletetramer, thus preventing its dissociation into αβ dimers. On the otherhand, the use of non-specific cross-linkers such as glutaraldehyde maylead to non-specific covalent bonding between amino acid residuesresiding within and between Hb tetramers. This leads to the formation ofhemoglobin polymers (polyHb) of various molecular weights and oxygenaffinities. Chemical reagents with multi-aldehyde functionalities can beused as cross-linking agents. These include molecules such asglutaraldehyde, ring-opened raffinose and dextran. In the case ofaldehydes, the formation of covalent cross-links may be initiated by thecarbonyl group of the aldehyde reacting with an amino group present inthe Hb tetramer. Polymerization of Hb into larger molecules may increasethe intravascular half-life of the polyHb with respect to nativetetrameric Hb and prevent Hb dissociation into αβ dimers. PolyHb may beeventually filtered out of the systemic circulation throughout thekidneys, the lymphatic system, and the reticuloendothelial system (RES).

Several other chemical agents can be used to cross-link hemoglobin α/βdimers and prevent their filtration by the glomerulus into the urine,and yet maintain the oxygen transport and delivery properties of nativehemoglobin. Bis 3′,5′ dibromo salicyl fumarate (DBSF) is an activateddiester of fumaric acid that has been used as a cross-linker tocross-link hemoglobin (Tye, U.S. Pat. No. 4,529,719, hereby incorporatedby reference in its entirety). Bis 3′,5′ dibromo salicyl fumarateeffects this change by associating the salicyl moieties with the sitesknown to bind aspirin within hemoglobin, and then effecting crosslinking by the fumarate active functionalities with the alpha and betachains. This maintains the two dimers in proper orientation forcross-linking with lysine residues. Cross-linking the α or β chains to alike chain of the other half of the tetramer forming hemoglobin canprevent disassociation of the tetramer and yields stable hemoglobins ofthe invention with a oxygen carrying capacity with a p50 of about 20 mmHg to about 45 mm Hg, with a p50 test performed in vitro in the absenceof CO₂. Cross linking is also possible between unlike chains in opposingdimeric pairs. Thus cross linking hemoglobin can address both the issuesof oxygen affinity, by locking the conformation of the modifiedhemoglobin into the T state, and the problem of rapid filtration by thekidney.

Hemoglobin's oxygen-binding capacity may be decreased in the presence ofcarbon monoxide because both gases compete for the same binding sites onhemoglobin, carbon monoxide binding preferentially relative to oxygen.Hemoglobin binding affinity for CO is 200 times greater than itsaffinity for oxygen, meaning that small amounts of CO may reducehemoglobin's ability to transport oxygen. When hemoglobin combines withCO, it forms a very bright red compound called carboxyhemoglobin. Wheninspired air (i.e., for example in the environment of tobacco smoking,cars, and furnaces) contains CO levels as low as 0.02%, headache andnausea may occur; if the CO concentration is increased to 0.1%,unconsciousness may follow. In heavy smokers, up to 20% of theoxygen-active sites can be blocked by CO. Hemoglobin also hascompetitive binding affinity for sulfur monoxide (SO), nitrogen dioxide(NO₂), nitric oxide (NO), and hydrogen sulfide (H₂S). The iron atom inthe heme group is in the Fe²⁺ oxidation state to support oxygentransport. Oxidation to Fe³⁺ state converts hemoglobin intomethemoglobin, which cannot bind oxygen. Nitrogen dioxide and nitrousoxide are capable of converting hemoglobin to methemoglobin.

Carbon dioxide occupies a different binding site on the hemoglobin.Hemoglobin can bind protons and carbon dioxide, causing a conformationalchange in the protein and facilitating the release of oxygen. Protonsbind at various sites along the protein and carbon dioxide binds at theα-amino group, hence forming carbamate. Conversely, when the carbondioxide levels in the blood decrease (i.e., around the lungs), carbondioxide is released, increasing the oxygen affinity of the protein. Thiscontrol of hemoglobin's affinity for oxygen by the binding and releaseof carbon dioxide is known as the Bohr effect.

As described above, the conformational change affected by the change inproton binding to hemoglobin facilitates oxygen offloading in tissueswhere the carbon dioxide concentration is increasing, with resultant pHdecrease. This creates a leftward shift of the cooperativity curve forhemoglobin's affinity for oxygen, yielding greater efficiency indelivery of oxygen per gram of hemoglobin. Enhancing this shift in amodified hemoglobin may result in an effective therapeutic interventionfor patients with poor cardiac function, thus providing more effectiveoxygenation with less work required by the heart. Additionally, ahemoglobin so modified to yield superior oxygen offloading can be usefulin treating patients subject to performance related oxygenationdeficits.

II. METHODS FOR PRODUCING CARBOXAMIDOMETHYLATED CROSS-LINKED HEMOGLOBIN

The steps for some of the embodiments of the present invention aredepicted in FIG. 1. Without limiting the scope of the present invention,the steps can be performed independently of each other or one after theother. One or more steps can be deleted in the methods of the presentinvention. The method of producing the hemoglobin of the presentinvention can include step 101 comprising removing plasma proteins andendotoxin from a preparation containing red blood cells by washing; step102 comprising lysing the red blood cells; step 103 comprisingseparating hemoglobin by removing stroma, including membranes andleucocytes, from the lysed red blood cells; step 104 comprising removingoxygen from the hemoglobin; step 105 comprising adding to a hemoglobinsolution a reagent which provides a thiol protecting group for acysteine of the hemoglobin; step 106 comprising separating thehemoglobin which has a thiol protecting group attached to a cysteinegroup of the hemoglobin; step 107 comprising cross linking thehemoglobin; and step 108 comprising equilibrating the hemoglobin inbiologicially compatible buffer and preparing a non-pyrogenic, endotoxinfree, oxygen free, stroma free, cysteine protected, cross linkedhemoglobin. Without limiting the scope of the present invention, theorder of the steps may be changed depending on the requirements forproducing a hemoglobin according to this invention.

1. Materials and Equipment Preparation

Whole blood from bovine sources may be obtained from live or freshlyslaughtered donors. Upon collection, the blood is typically mixed withat least one anticoagulant to prevent significant clotting of the blood.Suitable anticoagulants for blood are as classically known in the artand include, for example, sodium citrate, ethylenediaminetetraaceticacid and heparin. When mixed with blood, the anticoagulant may be in asolid form, such as a powder, or in an aqueous solution. It isunderstood that the blood solution source can be from a freshlycollected sample or from an old sample. The methods of the inventionprovide for the use of expired human blood from a blood bank. Further,the blood solution could previously have been maintained in frozenand/or liquid state. It is preferred that the blood solution is notfrozen prior to use in this method.

Prior to introducing the blood solution to anticoagulants, antibioticlevels in the blood solution, such as penicillin, may be assayed.Antibiotic levels may be determined to provide a degree of assurancethat the blood sample is not burdened with an infecting organism byverifying that the donor of the blood sample was not being treated withan antibiotic. Alternatively, a herd management program to monitor andinsure the lack of disease in or antibiotic presence from treatment ofthe cattle may be used. The blood solution may be strained prior to orduring the anticoagulation step, for example by straining, to removelarge aggregates and particles. A 150 micron filter is a suitablestrainer for this operation.

Any of a variety of assays may be employed to demonstrate thenon-pyrogenicity of the compositions of the present invention, forexample, but are not limited to, interleukin-6 and other cytokineinduction (Pool, E. J. et al., J. Immunoassay 19:95-111 (1998), and;Poole, S. et al., Dev. Biol. Stand. 69:121-123 (1988)); human monocytoidcell line assays (Eperon, S. et al., J. Immunol. Meth. 207:135-145(1997), and; Taktak, Y. S. et al., J. Pharm. Pharmacol. 43:578-582(1991)); the limulus amoebocyte lysate (LAL) test (Fujiwara, H. et al.,Yakugaku Zasshi 110:332-40 (1990), and; Martel F. et al., Rev FrTransfus Immunohematol 28:237-250 (1985)) and the rabbit pyrogen test(Bleeker W. K. et al., Prog Clin Biol Res 189:293-303 (1985); Simon, S.et al., Dev. Biol. Stand. 34:75-84 (1977), and; Allison, E. S. et al.,Clin. Sci. Mol. Med. 45:449-458 (1973)), all references incorporatedherein in their entirety. The rabbit pyrogen test was the preferredpyrogenicity assay until enhanced LAL-testing has replaced this formertechnique. It is understood that other methods of removing pyrogen areknown in the art and are within the scope of the present invention,including filters, absorbers, affinity materials, etc.

Serum lipases, such as lipase A, do not inactivate endotoxins bound tothe hemoglobin molecule. Therefore, endotoxins remain active toxins whentaken up by the hepatocyte metabolizing the hemoglobin. Friedman, H. I.et al. reported triad hepatoxicity in a rat model consistent with thistheory (See, Friedman, H. I. et al., Lab Invest 39:167-77 (1978), and;Colpan et al., U.S. Pat. No. 5,747,663) have reported a process forreducing or removing endotoxins from a cellular lysate solution.Wainwright et al. (U.S. Pat. No. 5,627,266) have described an endotoxinbinding protein immobilized to a solid support and the use of thismolecule in the removal of endotoxins from solution.

In some embodiments of the present invention, the elimination ofcontamination with endotoxins can be ensured by preventing theintroduction of endotoxins to the chemical processes of the presentinvention. Typically, endotoxins are added inadvertently by usingendotoxin contaminated water, non-sterile techniques, or the simpleprocess of bacteria exposure during collection. Measurement ofendotoxins can be difficult, and standard LAL binding assays do not workin the presence of hemoglobin since initial collection endotoxin bindsstrongly to hemoglobin. However, turbidometric, and chromogenic assayshave been validated that allow for very low limits of detection. Waterand the blood collection can be the most likely candidates forintroduction of endotoxins since increased number of steps in thepreparation of hemoglobin may increase the level of toxicity.Preparations using dialysis and filtration methods can expose thehemoglobin to a thousand volumes of water/buffer that may becontaminated with endotoxin. Membrane systems may be pretreated withNaOH or NaOCl to reduce or eliminate endotoxins. These materials maythen be flushed and cleaned from the various devices.

It is preferred that all membranes, and equipment used to produce thehemoglobin of the present invention be cleansed in a manner sufficientto cause the removal or elimination of endotoxin that may be present onsuch materials and equipment. Preferably, such cleansing is accomplishedby pre-washing surfaces and equipment that may come into contact withthe hemoglobin of the present invention using a dilute solution ofhemoglobin, previously qualified as non-endotoxin bearing. Such asolution serves to bind endotoxin and hence to remove residual endotoxinthat may be present on such membranes or equipment. See, for example,Tye, U.S. Pat. No. 6,894,150. The dilute solution of hemoglobin used forwashing is discarded after each use. Preferably, any ion removal orbuffer equilibration can be performed using counter flow dialysis so asto prevent accumulation of endotoxin in the subsequent product.2. Step 101. Washing of RBCs to remove Plasma Proteins and endotoxin.

The RBCs in the blood solution can be washed by any suitable means, suchas by diafiltration or by a combination of discrete dilution andconcentration steps with at least one solution, such as an isotonicsolution, to separate RBCs from extracellular plasma proteins, such asserum albumins or antibodies (e.g., immunoglobulins (IgG)). It isunderstood that the RBCs can be washed in a batch or continuous feedmode. Acceptable isotonic solutions are well known in the art andinclude solutions, such as, for example, citrate/saline solution or PBSwhich have a pH and osmolarity which does not rupture the cell membranesof RBCs and displaces the plasma portion of the whole blood. Sources ofpurified water which can be used in the method of invention includesdistilled water, deionized water (DI), water-for-injection (WFI) and/orlow pyrogen water (LPW). WFI, which is preferred, is deionized,distilled water. The specific method of purifying water is not asimportant as the requirement that it needs to be low in endotoxincontent.

The water and the reagents used in the present invention aresubstantially free from endotoxin contamination. Preferably, the waterand the reagents used in the present invention are completely free fromendotoxin contamination. One way to reduce the risk of endotoxincontamination can be to reduce the amount of water and reagent buffersexposed to the hemoglobin preparation. Therefore, under some embodimentsof the present invention, the hemoglobin preparations are made usingcounter-flow or counter-current dialysis for equilibration of buffersand/or removal of reaction products. Counter flow dialysis methods aresuitable for use in the present invention are commercially availablee.g., VariPerm M, bitop, Witten (see, e.g., Schwarz, T. et al,Electrophoresis 15:1118-1119 (1994)), Spectrum Laboratories, Inc.,Laguna Hills, Calif., etc. It is estimated that the hollow fibertechnique may yield a hemoglobin preparation of the present inventionthat has a 100 fold reduction in the amount of endotoxin as compared tostandard synthesis techniques. It is understood that other methods ofremoving the endotoxins are known in the art and are within the scope ofthe present invention.

In one method used to collect the erythrocytes, the blood samples can bewashed several times with an isotonic solution and the plasma can beseparated by centrifugation at 3,000 rpm in a 4″ diameter bowl.Preferably, the isotonic solution used is a saline solution. Preferably,the cells are washed at least three times, rinsed between eachcentrifugation, and resuspended in a final volume of an equal volume ofisotonic solution. Alternatively, concentration of RBCS may beaccomplished by filtration over a tangential flow membrane.

The use of a sonicator may be discouraged as it makes membrane spheres(often referred to as “dust”). Agitation methods suitable for use in thepresent invention may include a magnetic stir bar (0.25″ in diameter)and a mechanical rocker or shaker. (one to two liter container capacitymay be used). This exemplary protocol describes equipment to illustratethe limitation of forces acting upon the collected cells to preventundesirable fracturing of the cell membranes at this point.

It is understood that methods generally known in the art for separatingRBCs from other blood components can be employed. For example,sedimentation, wherein the separation method does not rupture the cellmembranes of a significant amount of the RBCs, such as less than about30% of the RBCs, prior to RBC separation from the other blood componentssuch as, white blood cells (WBCs) and platelets.

White blood cells can cause febrile reactions in human recipients whenpresent in transfused packed RBCs. It is desirable to use aleucoreduction filter which can pass the RBCs but markedly reduce thenumber of WBCs. A larger prototype than that used for single human unitof packed cells is used to evaluate the leucoreduction. Prechillingwashed bovine erythrocytes for about 12 h permits leukoreduction offiltration in about 15 minutes. The results are shown in Table 1. A 3log reduction in WBCs, as quantified by instruments such as a CoulterCounter® Cell and Particle Counter is achieved by the passage of the redcell suspension through a leucocyte reduction filter. This is analternative to the method wherein RBCs are selectively lysed in thepresence of WBCs without lysing the WBCs, which are subsequently removedby filtration. This selective lysing is discussed more fully below.

TABLE 1 Leucocyte Reduction Filter Initial Vol Adjusted % Sample WBC/mm³Final WBC/mm³ Removal Log₁₀ Removal 12 h Cold 6.13 × 10³ 28 99.6% 3Bovine

3. Step 102. Lysis of Erythrocytes.

Various lysis methods can be used, such as mechanical lysis, chemicallysis, hypotonic lysis or other known lysis methods which releasehemoglobin without significantly damaging the ability of the Hb totransport and release oxygen. Hemoglobin may be released from theerythrocyte by hypotonic lysis in deionized water. Preferably, lysis isaccomplished in four to twenty volumes of deionized water. In onemethod, plasma free blood cells are equilibrated with NS, and thendiluted into 4 volumes of deionized water (DI). This can result in thefracturing of the plasma free blood cells by the hypotonic lysis. Thecells are fractured by the rapid uptake of water. Red blood cells can belysed in about 30 seconds, while WBCs are more resistant. RBCs arecollected in a flow process after the RBCs are allowed to lyse but justbefore the WBCs begin to lyse, an additional volume of a 9% salinesolution is added to arrive at a total concentration of 0.9% salinecontent overall. This timed increase in salinity prevents WBCs fromlysing. The stroma and WBCs are removed from the lysed RBCs byfiltration. The hemoglobin can then be removed by a 0.22 μm filter asfiltrate while the retentate would concentrate the stroma, red cellmembranes, and the unlysed WBCs. FIG. 2 depicts the time course of alysis experiment showing resistance of WBC lysis for up to 5 minutes.Erythrocyte lysis can be stopped during the two minute period beforeappreciable leucocyte lysis occurs.

Other methods of erythrocyte lysis, such as “slow hypotonic lysis” or“freeze thaw”, may also be employed. See, e.g., Chan et al., J. CellPhysiol. 85:47-57 (1975), incorporated by reference in its entirety. Insome embodiments of the present invention, the cells are lysed by flowmixing red blood cells in isotonic saline with 12 volumes of deionized,endotoxin-free water and subjecting the cells to gentle agitation. It isunderstood that other methods of lysing the RBCs are known in the artand are within the scope of the present invention.

4. Step 103. Separation of Stroma from Hemoglobin.

The contents of the erythrocyte are about 98.5% in pure hemoglobin, withsome small amount of other proteins including carbonic anhydrase. Themembranes of red blood cells are referred to as ghosts or stroma andcontain all of the blood type antigens. Rabiner et al. firstdemonstrated that some of the toxic properties of hemolyzed red bloodcells were related to the membrane (stroma) of red blood cells and theirrelated lipids (Rabiner et al., J. Exp. Med. 126:1127 (1967),incorporated by reference in its entirety). The membranes can bedestroyed by freezing so that storage requirements for blood may requireclimate controlled refrigeration. In addition, many of the human viraldiseases transmitted through blood transfusions may adhere to the stromaof red blood cells. Thus, stroma-free hemoglobin (“SFH”) can bebeneficial in light of the immunogenic properties, such as inflammation,agglutination, clotting, an immune mediated complement response,platelet activation, etc, of the cell membranes of red blood cells, andpossibility of viral contamination.

An effective stroma-free hemoglobin blood substitute or oxygen deliverytherapy can offer several advantages over conventional blood basedtherapies. Significantly, the use of stroma-free hemoglobin bloodsubstitutes can reduce the extent and severity of undesired immuneresponses, and the risk of transmission of viral diseases, includinghepatitis and HIV. Moreover, in contrast to the limited storage capacityof erythrocytes, a stroma-free hemoglobin blood substitute or oxygendelivery therapeutic can exhibit an extended shelf life, and requireless rigorous environmentally controlled storage facilities.

The stroma may be removed by ultrafiltration of the hemolysate over a0.65 micron filter which retains the cellular components and passes thehemoglobin. Alternatively, the cellular debris may be removed bysubsequent filtration through a 0.22 micron filter or a 300,000 Daltonmolecular weight filter. Ultrafiltration membranes suitable for use inthe present invention are commercially available from, for example,Millipore Corporation. Other methods for separating Hb from the lysedRBC phase can be employed, including sedimentation, precipitation (Tye,U.S. Pat. No. 4,529,719), centrifugation or microfiltration It isunderstood that other methods of removing stroma are known in the artand are within the scope of the present invention.

Carbonic Anhydrase.

Carbonic anhydrase will be removed through diafiltration once the redcell membrane has been lysed, which is employed at several points inthis method. For example, diafiltration and buffer exchanges occurbefore, during and after cross-linking. The presence of carbonicanhydrase may be quantified by ELISA.

Microscopic analysis of 10 ml spun samples does not reveal any cellulardebris.

Phospholipid Level Reduction.

Another key element to the stable NO-blocked tetrameric hemoglobins ofthe invention is the low level of phospholipids present. Phospholipidsderive from the surface lipid layer of the red cells, the source of thehemoglobin. The steps of processing given above remove these unwantedlipids, thus eliminating the problems associated with their presence.Phospholipid assays can be measured by HPLC and/or ELISA as is wellknown to one skilled in the art. Phosphatidylcholine is found to bebelow the limit of detection.

Concentration of Hemoglobin to 14% Solution.

After such treatment, the stroma-free hemolysate is concentrated by amembrane that does not allow for the passage of hemoglobin. Preferably,the stroma-free hemolysate is concentrated using a filter having a10,000 MW cut-off. Preferably, the stroma-free hemolysate isconcentrated to a 1%-25% (g/1) solution. More preferably, thestroma-free hemolysate is concentrated to about 5% to about 20%. Mostpreferably, the stroma-free hemolysate is concentrated to about 6% toabout 10%. The concentrated solution can be equilibrated with buffer andthe pH is adjusted. Preferably, the pH is adjusted to a pH of 7.40.However, a pH of between about 6.5 and about 8.5 can be used in thepresent invention.

Optionally, the concentrated Hb solution can then be directed into oneor more parallel chromatographic columns to further separate thehemoglobin by high performance liquid chromatography from othercontaminants such as antibodies, endotoxins, phospholipids and enzymesand viruses. Examples of suitable media include anion exchange media,cation exchange media, hydrophobic interaction media and affinity media.The chromatographic columns may contain an anion exchange mediumsuitable to separate Hb from non-hemoglobin proteins. Suitable anionexchange mediums include, for example, silica, alumina, titanium gel,cross-linked dextran, agarose or a derivatized moiety, such as apolyacrylamide, a polyhydroxyethyl-methacrylate or a styrenedivinylbenzene, that has been derivatized with a cationic chemicalfunctionality, such as a diethylaminoethyl or quaternary aminoethylgroup. A suitable anion exchange medium and corresponding eluents forthe selective absorption and desorption of Hb as compared to otherproteins and contaminants, which are likely to be in a lysed RBC phase,are readily determinable by one of reasonable skill in the art.

Removal of Phosphate Ion.

Bucci et al. (U.S. Pat. No. 5,290,919) have reported that removal oforganic phosphates, e.g., 2,3-diphosphoglycerate, may be necessary inhuman hemolysates because the site of the cross-linking reaction is thesame as that occupied by 2,3-diphosphoglycerate in hemoglobin. In someembodiments of the present invention, the stroma free human Hb solutionis substantially free from inorganic phosphate. Accordingly, in someembodiments of the present invention, the stroma free human Hb (beforecross linking) that has passed through the filter may be then treated toexchange phosphate for chloride. For this purpose, the stroma free humanHb can be passed in the absence or presence of oxygen, through an ionexchange column that has been previously prepared and equilibrated withchloride. Efficacy of this step may be measured by total inorganicphosphate analysis. Suitable ionic resins are commercially available andare within the scope of the present invention. The ionic resin removesphosphate that may compete for the site to which aspirin binds duringthe reaction with DBSF. The solution can then be concentrated to thedesired range. This operation is not necessary when using bovine Hb.

5. Step 104. Removal of Oxygen.

The thiol blocked stroma free Hb or more specifically, the CMSFH can betreated under conditions sufficient to remove oxygen present in thepreparation. One aspect of the present invention concerns an improvedprocess for removing oxygen from CMSFH preparations. Without limitingthe scope of the present invention, such deoxygenation can be carriedout before or after any of the steps disclosed herein. For example, thedeoxygenation step can be performed prior to or after the step ofremoving stroma, the step of removing the endotoxins, the step of thiolprotection, the step of phosphate removal, the step of lysis of RBCs, orthe step of cross-linking of the hemoglobin. In one embodiment, suchdeoxygenation is performed prior to the protection of the thiol group inthe cysteine moiety of the hemoglobin of the present invention. Inanother embodiment of the invention, deoxygenation is performed prior tocross linking the hemoglobin. In some embodiments of the invention, thesteps of the method may require more time to be completed. In suchcases, deoxygenated conditions may be preferred.

The extent of deoxygenation can be measured by gas chromatograph,zirconium-based detector (e.g., a “MOCON” analyzer (Mocon, Minneapolis,Minn.), by measuring pO₂ or by measuring the spectral shift that ischaracteristic of deoxyhemoglobin formation.

Oxygen in the hemoglobin can be removed by vacuum, or by vacuumcentrifugation. The CMSFH used may be an ultrafiltrate obtained from theremoval of stroma (dilute) or a retentate from the ultrafiltration ofthe second stage ultrafiltration conducted to concentrate the hemoglobinto approximately 10% (w/v). Either of these solutions of CMSFH obtainedcan be readily deoxygenated by applying a vacuum sufficient to equal thepartial pressure of water at the temperature of the solution, while thesolution can be centrifuged at a speed sufficient to produce a forcegreater than the surface tension of the solution. These are generallylow speeds and can be met with preparatory centrifuges, or those of acontinuous flow variety. It may be desirable to consider the geometry ofthe containers of the CMSFH to insure that there may be adequate surfacearea for gas exchange and that the temperature can be maintained and thesolution not allowed to freeze.

Contactor membrane technology can be used to remove O₂ from Hbsolutions. The contactor membrane technology can also be used foroxygenation where oxygen gas may be used instead of nitrogen gas. Threeor four of such membranes may be attached in series for higherthroughput and can be used for commercial production of deoxygenated oroxygenated hemoglobin solution. The Hb concentration affects the rate atwhich the dissolved O₂ is removed. As the Hb concentration is loweredthe O₂ removal rate increases. The experiment may not lower O₂concentration to <100 ppb. However, the test can be performed in theanaerobic glove box to make the system gas tight. The glove box canmaintain the environment at very low O₂ levels (<5 ppb). This glove boxenvironment can provide the O₂ barrier required to ensure that no O₂ canbe re-absorbed by the Hb. Hg vacuum greater than 28.5″ (<50 mm Hg) canbe used for optimum O₂ removal.

The deoxygenated, endotoxin free, stroma free, carboxamidomethylated Hb(dCMSFH) prepared in the manner described above may be preferablymaintained in an inert environment and the pH of the preparation may bepreferably adjusted to a range between 6.0 and 9.5, and most preferablyabout pH 8.3-8.4. The pH of the solution may be adjusted using 1.0 Nacetic acid or 1.0 N NaOH. Where dilution, suspension, or addition ofwater (including buffers, etc.) for other purposes is desired, suchwater may be deoxygenated and be free of endotoxin. All subsequent stepsmay be carried out in the absence of oxygen, maintained by what evermeans is desired. As indicated above, a preferred method involves theuse of nitrogen positive pressure environmental glove box, however,other inert gases (e.g., argon) may be equivalently employed in lieu ofnitrogen.

6. Step 105. Protecting the Sulfhydryl of the Cysteine(s) of theHemoglobin.

The step of protecting cysteine of the hemoglobin with thiol protectinggroups may be carried out before the cross linking step or after thecross linking step. In one embodiment of the present invention, the stepof protecting the thiol group in the cysteine moiety is carried outbefore the cross-linking step. In another embodiment of the presentinvention, the step of deoxygenating the hemoglobin is carried outbefore the step of protecting the thiol group in the cysteine moiety. Insome embodiments of the invention, deoxygenation is not performed priorto protecting the sulfhydryl groups in the hemoglobin. All the reagentsknown in the art for the protection of a functional group such as, butnot limited to, hydroxyl, thiol, or carboxyl, are included in thepresent invention.

Some of the examples of the reagents include, but are not limited to,4-pyridylmethyl chloride, alkoxyalkylchloride, dimethoxymethane,N-(hydroxymethyl)acetamide, triphenylmethyl chloride, acetyl chloride,2-chloroacetic acid, acetic anhydride, haloacetamide such as,iodoacetamide, bromoacetamide, chloroacetamide, or fluoroacetamide,haloacetate such as iodoacetate, bromoacetate, chloroacetate, orfluoroacetate, benzyl chloride, benzoyl chloride, di-tert-butyldicarbonate, p-hydroxyphenacyl bromide, p-acetoxybenzyl chloride,p-methoxybenzyl chloride, 2,4-dinitrophenyl fluoride, tetrahydropyran,acetamidohydroxymethane, acetone, bis-carboethoxyethene,2,2,2-trichloroethoxycarbonyl chloride, tert-butoxycarbonyl chloride,alkyl isocyanate, and alkoxyalkyl isocyanate. In a specific example ofthe sulfhydryl protected hemoglobins of the invention, the reagent isiodoacetamide. It is understood that any reagent known in the art thatcan be used for carboxamidomethylation.

Optimization of the Reaction with Iodoacetamide (IAM):

The iodoacetamide reaction is followed with an iodide specificelectrode, since one of the byproducts is iodide ion. A two molar excessper equivalent of sulfhydryl group can be used. Table 2 shows a grid oftime course of the iodoacetamide reaction for bovine Hb vs. the moles ofIAM reagent used in the IAM reaction. The results show the amount offree sulfhydryl per mole of Hb and are given in units of molarequivalents relative to bovine Hb.

TABLE 2 Reaction of Bovine Hb with IAM. Results given in equivalents offree sulfhydryl remaining. Moles Time IAM 0 15 30 45 60 75 90 120 2 21.0 0.5 0.5 4 0.5 0.2 0.1 <0.17. Step 106. Separating Thiol-Protected Hemoglobin from Reactants.

After the reaction is complete, as determined by the rate of iodideevolution observed, excess IAM is removed by equilibration with Ringer'sAcetate and diafiltration.

8. Step 107. Cross-Linking with DBSF and Reaction with PLP.

Stroma-free Hb can be prevented from dissociation into α, β dimers bycross-linking intramolecularly to prevent dissociation of the tetramerinto α, β dimers and thus increase its circulatory half-life. Thisrestricts Hb into the T state and resultantly can modify the affinityfor oxygen and therefore modifies the oxygen transport properties of theHb.

Examples of suitable cross-linking agents include polyfunctional agentsthat will cross-link Hb proteins, such as glutaraldehyde,succindialdehyde, activated forms of polyoxyethylene and dextran,α-hydroxy aldehydes, such as glycolaldehyde,N-maleimido-6-aminocaproyl-(2′-nitro,4′-sulfonic acid)-phenyl ester,m-maleimidobenzoic acid-N-hydroxysuccinimide ester, succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate,m-maleimidobenzoyl-N-hydroxysuccinimide ester,m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester,N-succinimidyl(4-iodoacetyl)aminobenzoate,sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl4-(p-maleimidophenyl)butyrate, sulfosuccinimidyl4-(p-maleimidophenyl)butyrate,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,N,N-phenylene dimaleimide, and compounds belonging to the bis-imidateclass, the acyl diazide class or the aryl dihalide class, among others.The saccharides can be used as cross-linking agents. The examples ofsaccharides include, but are not limited to, monosaccharides (galactose,glucose, methylglucopyranoside, and mannitol), disaccharides (lactose,maltose, cellobiose, sucrose, and trehalose), a trisaccharide(raffinose) and polysaccharides (dextrans with molecular weights of15,000 and 71,000 Da).

The cross-linking of hemoglobin may be conducted in the absence ofoxygen. Inorganic phosphate, which binds tightly to the hemoglobinmolecule and interferes with the cross-linking reaction, may be removedto increase yield. Endotoxins, which bind tightly to the hemoglobinmolecule and become a hepatic toxin when the hemoglobin is metabolized,may not be allowed to contact the hemoglobin.

A suitable amount of a cross-linking agent may be that amount which maypermit intramolecular cross-linking to stabilize the Hb and alsointermolecular cross-linking to form polymers of Hb, to thereby increaseintravascular retention. Without limiting the scope of the presentinvention, various strategies can be employed to cross-link Hb withdesirable molecular weight distributions and oxygen binding properties.The type and concentration of both cross linking and quenching agents,the duration of the cross-linking/polymerization reaction, andutilization of reducing agents are all possible variables that can bemodified in these reactions to engineer the molecular weightdistribution, oxygen binding properties, and metHb levels ofcross-linked Hb dispersions.

Optimization of Cross Linking Using pH Control and Excess Cross LinkingAgent:

Table 3 shows a grid of increasing pH vs. mole ratio of cross linkingagent (DBSF) over 2 h. The results show the percentage of alpha chainleft unreacted at different pH and equivalents (in moles) DBSF at theend of the 2 h period. The pH is maintained constant with titration ofthe acid produced by reaction with NaOH. After 2 h, the production ofacid has long since ceased. Results of cross linking are determinedusing gel electrophoretic separations, looking for residual uncrosslinked alpha chains. In the standard method of production of the stableNO blocked tetrameric hemoglobins of the present invention, using 2equivalents of DBSF, better than 98% cross-linking is achieved.

TABLE 3 pH vs. Molar Ratio DBSF. Percentage of Uncross-Linked HemoglobinRemaining. Equivalents pH DBSF 7 7.5 8 8.2 8.4 1  21% 1.2  19% 1.5   3%2.0 38% 11% 2.5% 1.7% 1.5% 2.5 1.7%

In some embodiments of the present invention, the dCMSFH is cross-linkedwith bis 3′,5′ dibromo salicyl fumarate (DBSF) (Tye, U.S. Pat. No.4,529,719, hereby incorporated by reference in its entirety). DBSFcross-linker may be added with stirring to the dCMSFH preparation at amolar ratio of DBSF cross-linker:dCMSFH of greater than 1:1. Preferably,the molar ratio of DBSF cross-linker:dCMSFH is 2:1. Prior to suchaddition, the pH of the dCMSFH preparation is adjusted to 8.4 andmaintained at 8.4 throughout the reaction. The pH of the reactionmixture is carefully maintained by the addition of acid or base sincethe solution is not buffered. The reaction is permitted to go tocompletion.

Determination of Extent of Cross Linking Using SDS Gel Electrophoresis:

SDS can denature proteins to form long rods covered by negative chargesof the carboxyl group at neutral pH. Proteins can then be separated bysize exclusion using electrophoresis since the manifold excess ofnegative charge by the SDS can dwarf the charge heterogeneity of thenative proteins. The Beckman Coulter PA-800® provides rapid record ofthe gel electrophoresis by using the absorption at 216 nm for thepeptide bond. Standard protein mixtures can be used to calibrate thecolumn for the range of molecular weights of interest, between 10 KDaand 100 KDa in the present case, as shown in FIG. 3.

Native hemoglobin run on SDS gel electrophoresis can give two peaks withalmost baseline separation between them. The first peak has been shownto be the alpha chain and the second the beta chain of hemoglobin. Theycan occur in almost equal amounts as there are equal numbers of chainsin the molecule. This is illustrated in FIG. 4A, which is an expandedregion of the overlaid electropherograms shown in FIG. 4B. Nativehemoglobin appears as the green trace (rt13.98 and rt14.14), crosslinked hemoglobin according to the present invention (dXCMSFH) appearsas the red trace, and size standards are blue. The overlaidelectropherograms shown in FIG. 4A are slightly offset for ease ofviewing. The expanded region illustrated is clustered about the 20 kDasize standard, which is the region of interest for the two alpha andbeta subunits of native hemoglobin. The experiment shown here for thecross-linked material is taken from a midpoint in the process, when mostof the alpha chains have already cross linked, but significant amountsof beta chain still remain unreacted.

In FIG. 4B, the overlay of the full width electropherograms are shown,again with native hemoglobin in green, the cross linking experiment inred, and the size standards in blue. The fumaryl cross linking of thetwo alpha chains yields a pair of peptides tethered together and thuswill appear at later elution times than the unreacted beta chains. Theseries of three new major product peaks at a higher molecular weight ofabout 36,000-45,000 KDA are seen in FIG. 4B at rt of 16 to 17 min. Theproduct peaks may not be quantified as the size standards are all singlepeptide chains and cannot be extended for quantification for thedimerized products of this reaction. However, it can be seen that threehigher molecular weight products are being formed.

There is a formation of β-β crosslink's in addition to the α-αcrosslink's. There is a small amount of material at 90,000 KDa which canindicate the formation of a small number of inter molecular crosslink's.

Reaction of Human Hb with PLP.

Pyridoxal-5-phosphate (PLP) has the ability to modify many hemoglobins.Although the properties of dXCMSFH from human hemoglobin benefit fromthe pyridoxal-5-phosphate reaction, dXCMSFH from bovine hemoglobin doesnot require this step. PLP modifies human hemoglobin by introducing anegative charge near a penultimate β chain histidine residue and byremoving a positive charge at the amino terminal end of the same chain.These charge changes stabilize a new molecular configuration that issimilar to the hemoglobin-DPG (diphosphoglycerate) complex.Significantly, the hemoglobin of this new configuration has an oxygenaffinity resembling that of native hemoglobin within the red cell. Theproduct may have one or two PLP molecules attached per tetramer. Inprior PLP-hemoglobin preparations the intravascular retention time wastoo short to permit such preparations to be acceptable as aresuscitation fluid. Additionally, they were found to cause osmoticdiuresis.

Accordingly, after the cross-linking reaction has been completed, whereusing human Hb, pyridoxal-5-phosphate (PLP) is added to thedeoxygenated, endotoxin free, stroma free, carboxamidomethylatedcross-linked human Hb (dXCMSFH) preparation. The PLP is reacted with thedXCMSFH and then reduced with sodium borohydride to formdXCMSFH-pyridoxal-5′-phosphate (dXCMSFH-PLP) using the methods describedby Benesch et al. (Benesch et al., Biochemistry 11:3576 (1972) andreferences therein; Benesch et al. Proc. Natl. Acad Sci. 70 (9): 2595-9(1974); Benesch et al., Biochem. Biophys. Res. Commun. 63(4): 1123-9(1975); Benesch et al., Methods Enzymol. 76:147-59 (1981); Benesch etal., J. Biol. Chem. 257(3):13204 (1982); Schnackerz et al.; and, J.Biol. Chem. 258(2):872-5 (1983), all of which references areincorporated herein by reference in their entirety) with the change thatall reagents are free of endotoxin and oxygen and the reaction occurs inthe absence of oxygen. This treatment is not necessary when using bovineHb

Determination of Residual Uncross-Linked Hemoglobin.

High performance liquid chromatography (HPLC) Size ExclusionChromatography (SEC) can be used to determine the percent of totalcross-linked Hb, percent of cross-linked tetramer, or percent of crosslinked higher order species of Hb/polyHb dispersions. A salt such as,MgCl₂ can serve to dissociate any non-cross-linked tetrameric Hb intoα-β dimers, while cross-linked tetrameric Hb may remain intact. Hence,non-cross-linked Hb may elute in a separate peak away fromintramolecularly cross-linked Hb.

Size exclusion HPLC on a Biorad Bio-Sil^(R) SEC-12.5-5 column of thereaction mixture using 0.5M MgCl₂ solution as a buffer under conditionsthat would otherwise not denature the hemoglobin secondary structure,may be used to examine the amount of unreacted material, since underthese conditions the equilibrium would favor the alpha beta dimer with amolecular weight of 32 KDa, which would be expected at greater retentiontimes than seen for any peak in this experiment. As seen in FIG. 5, theHPLC trace shows the major peak at 64 KDa, with a minor peak at 128 KDa.There is no material at later elution times, and hence no materials withlower molecular weight. This experiment demonstrates the completeabsence of unreacted hemoglobin and illustrates that most of thematerial is the stabilized tetramer of Hb with a molecular weight of 64KDa.

9. Step 108. Preparing Hemoglobin Solutions by Equilibration.

The deoxygenated stable NO blocked tetrameric Hb and, in particular,dXCMSFH can be equilibrated with Ringer's lactate or Ringer's acetatesolution, which under conditions of diafiltration, removes excess DBSFand byproducts of the reaction, for example, dibromosalicylic acid.Preferably, any ion removal or buffer equilibration can be performedusing counter flow dialysis so as to prevent accumulation of endotoxinin the subsequent product. After equilibration, the solution can besterile filtered into suitable infusion containers. Infusion containerssuitable for use in the present invention may include, but are notlimited to, sterile IV bags. Preferred infusion containers may preventgas exchange (i.e., impermeable to oxygen) and the dXCMSFH can be storedin the absence of oxygen. This is expected to prevent heme oxidationwhich forms methemoglobin.

Determination of the Affinity for Oxygen by Modified Hemoglobin.

Hemoglobin has an ability to bind and release oxygen under physiologicalconditions as a function of the partial pressure of oxygen in thesystem. Oxygen affinity of the hemoglobin derivative of the presentinvention can be measured using the Hemox-Analyzer (made by TCSCorporation or the gill cell described by Dolman et al., Anal. Biochem.87:127 (1978), incorporated by reference in its entirety.

Hemox-Analyzer (made by TCS Corporation) allows the determination of thehemoglobin oxygen dissociation curve. Other methods to obtain ahemoglobin oxygen dissociation curve may not be as reproducible,accurate and easy to perform. The hemoglobin oxygen dissociation curvescan be altered by changes in pH, temperature, CO₂ concentration, speciesof hemoglobin, variant of human hemoglobin, hemolyzed hemoglobin, andthe like. The shape of the curve and the shift of the curve along the Xaxis can describe the ability of the hemoglobin to load and unloadoxygen. The information can be useful in research for blood and modifiedhemoglobin as it is an in vitro test of in vivo function. It can measurethe ability of hemoglobin to load and unload oxygen. A shorthanddescription of the entire hemoglobin dissociation curve can be given bythe p50 for O₂, the partial pressure of oxygen in mm of Hg, which cancause the hemoglobin to be half saturated with oxygen.

Chemical modifications to hemoglobin or genetic variants to hemoglobincan cause the p50 to decrease, i.e. bind oxygen more tightly at anygiven oxygen pressure. In vivo this can mean less oxygen to perfusedtissue.

The Hemox-Analyzer relies upon the change in color of blood (hemoglobin)that is arterial (oxygenated, red) and venous (less oxygenated, blue),and an oxygen electrode. A small dilute sample is prepared in a specialspectrophotometric cell that has a small orifice in the bottom thatallows a purified gas to be slowly bubbled through a stirred solutionand also fitted with an oxygen electrode. The entire cell is preciselytemperature controlled at 37° C., to equilibrate to body temperature.The outputs of the spectrophotometer and the oxygen electrode areanalyzed and plotted. At the beginning of a plot the sample is fullyoxygenated by bubbling pure oxygen through the sample until the oxygensaturation is greater than the fraction of oxygen in air (21%), then thegas bubbler is switched to nitrogen and the removal of oxygen begins.Oxygen equilibrium curves can thus be generated.

The p50 for human hemoglobin in RBCs can be about 28. The p50 falls toabout 14 when RBCs are separated from the 2,3DPG which forms a saltbridge in the red cell to decrease the oxygen affinity. The p50 forbovine hemoglobin whether in the red cell or in free solution is about25 to about 40 depending on the pH and the concentration of CO₂. FIG. 6shows oxygen affinity curves for bovine whole blood, stroma free Hb,cross linked dXCMSFH of the invention, and fresh human blood. In thecross linked hemoglobin, the cross linking locks the hemoglobin in thetense state and therefore loses the sigmoidal curve. At lower pO₂, whenO₂ is delivered to the tissues, the cross linked hemoglobin deliversmore oxygen as compared to bovine whole blood, stroma free Hb, and freshhuman blood. The p50 of cross linked hemoglobin is higher than that ofhuman hemoglobin, and bovine hemoglobin. The p50 value for bovine wholeblood is 24.73 mm Hg, for stroma free Hb is 21.20 mm Hg, for fresh humanblood is 23.72 mm Hg and for dXCMSFH is 32.43 mm Hg, which issignificantly higher than that of human RBCs. Therefore, the stableNO-blocked tetrameric Hb of present invention, and in particular,XCMSFH, may demonstrate greater efficiency to the delivery of oxygen pergram of hemoglobin. Although these numbers, as measured herein, are notidentical to literature values (i.e., human blood p50 literature valueis 27 mm Hg compared to 23.72 mm Hg reported here), the present valuesare a good relative measure of oxygen offloading performance.

The deoxygenated, endotoxin free, stroma free, carboxamidomethylatedcross-linked Hb (dXCMSFH) of the present invention is a stabilizedtetramer of bovine hemoglobin that is locked in the tense or T state,and has a p50 similar to hemoglobin within normal human red blood cellsor, as shown in FIG. 6, higher. Thus, an equal amount of hemoglobin froma human red blood cell and hemoglobin from dXCMSFH, can carry the sameamount of oxygen leaving the lung. However, dXCMSFH can deliver slightlymore oxygen before its venous return, based upon the data shown in FIG.6.

III. ANALYSIS 1. Physical Characteristics of the Stable NO-BlockedTetrameric Hemoglobins of the Invention

A stable NO-blocked tetrameric hemoglobin of the present invention has amolecular weight distribution of about 65 kDa, a p50 of 20-45 mm Hg, anosmolality of 290-310 mOsm/Kg, with a pH of 6.0 to 7.9 at 10-22° C. Themodified hemoglobins of the invention have a total hemoglobin of 6.0-20g/dL, with methemoglobin levels of less than or equal to 5%,oxyhemoglobin levels of less than or equal to 10%. The modifiedhemoglobins of the invention have endotoxin levels of less than or equalto 0.02 EU/ml, phosphatidylcholine levels below detection limits, meettest for sterility, and a low level of extraneous organics. The modifiedhemoglobins of the present invention have a sodium ion level of 125-160mmol/l, a potassium ion level of 3.5-5.5 mmol/l, a chloride ion level of105-120 mmol/l, and a calcium level of 0.5-1.5 mmol/l. The modifiedhemoglobins of the invention have levels of N-acetyl cysteine of lessthan or equal to 0.22%.

2. Analytical Methods Physical Chemical Analysis

The deoxygenated stable NO blocked tetrameric Hb and dXCMSFH, inparticular, as disclosed herein can be analyzed at any step of theprocess of making it. Hemoglobin can be analyzed, for example, but notlimited to, after removing stroma, after removing endotoxin, afterlysis, after removing oxygen, after protection of thiol group in thecysteine moiety, or after cross-linking etc. The hemoglobin can beanalyzed for purity, absorbance, structure, p50, nitric oxide bindingcapacity, white blood cell (WBC) count, microorganism growth in thehemoglobin solution, cross-linking, amino acid analysis, proteinanalysis, or effect of refrigeration or storage. Various analyticaltechniques are known in the art and are all within the scope of thepresent invention. Some of the examples of the analytical techniques areprovided herein but they are not limiting to the scope of the presentinvention.

A. Mass Spectrometry (MS).

There are many types of mass spectrometers and sample introductiontechniques which allow a wide range of analyses and they are allincluded herein. In some preferred embodiments of the present invention,the technique used is mass spectrometry. Mass spectrometers may consistof three distinct regions: Ionizer, Ion Analyzer, and Detector.Ionization methods include, but are not limited to, electron impact(EI), chemical ionization (CI), electrospray (ESI), fast atombombardment (FAB), and matrix assisted laser desorption (MALDI).Analyzers include but are not limited to, quadrupole, sector (magneticand/or electrostatic), time-of-flight (TOF), and ion cyclotron resonance(ICR). Other related techniques are, for example, ion mobilityspectrometry/mass spectrometry (IMS/MS), Tandem mass spectrometry(MS/MS), Orbitrap mass spectrometry. FTICR mass spectrometry,single-stage or a dual-stage reflectron (RETOF-MS, ladder sequencingwith TOF-MS), Post-source decay with RETOF-MS MALDI, In-source decaywith linear TOF-MS, and surface-enhanced laser desorptionionization-time of flight (SELDI-TOF). The mass spectrometer may becoupled with LC or GC.

B. UV-Vis.

In some embodiments of the present invention, optical absorptionspectroscopy (UV/VIS) has been used to determine the absorbance rangefor the hemoglobin. UV/VIS plays a role for the determination ofconcentrations of macromolecules such as proteins. Organic dyes can beused to enhance the absorption and to shift it into the visible range(e.g. Coomassie blue reagents). Understanding the forces that govern theinteraction of proteins with one another assists in the understanding ofsuch processes as macromolecular assembly, chaperone-assisted proteinfolding and protein translocation. Resonance Raman spectroscopy (RRS) isa tool which can be used to study molecular structure and dynamics.Resonance Raman scattering requires excitation within an electronicabsorption band and results in a large increase of scattering. Thisapproach may help to investigate specific parts of macromolecules byusing different excitation wavelengths.

C. Liquid Chromatography (LC).

Liquid chromatography is a tool for isolating proteins, peptides, andother molecules from complex mixtures. In some embodiments of thepresent invention, LC has been used for separation, purification andanalysis of the hemoglobin and excipients used in the formulations ofthe invention. Examples of LC include affinity chromatography, gelfiltration chromatography, anion exchange chromatography, cationexchange chromatography, diode array—LC and high performance liquidchromatography (HPLC) and affinity and size exclusion chromatographyHPSEC.

Gel filtration chromatography and HPSEC chromatography separatesproteins, and peptides on the basis of size. Gel FiltrationChromatography may be used for analysis of molecular size, forseparations of components in a mixture, or for salt removal or bufferexchange from a preparation of macromolecules.

Affinity chromatography is the process of bioselective adsorption andsubsequent recovery of a compound from an immobilized ligand.

Ion exchange chromatography separates molecules based on differencesbetween the overall charges of the proteins. It is usually used forprotein purification but may be used for purification of peptides, orother charged molecules. Elution can be achieved by increasing the ionicstrength to break up the ionic interaction, or by changing the pH of theprotein.

HPLC can be used in the separation, purification and detection ofhemoglobin of the present invention. Use of reversed-phasedchromatography (RPC) can be utilized in the process of protein structuredetermination. The normal procedure of this process can be 1)fragmentation by proteolysis or chemical cleavage; 2) purification; and3) sequencing. A common mobile phase for RPC of peptides can be, forexample, a gradient of 0.1% trifluoroacetic acid (TFA) in water to 0.1%TFA in a suitable organic solvent, such as acetonitrile, which providesfor the solubilization of the proteins/peptides, permits detection atapproximately 230-240 nmm, and is easily removable, i.e by evaporation,from the proteins/peptides.

The use of size-exclusion chromatography (SEC) and ion-exchangechromatography (IEC) can be used in determining the structure of thehemoglobin of the present invention. Full recovery of activity afterexposure to the chromatography may be achieved, and SEC columns canallow fractionation from 10 to 1000 kilodaltons. The use of gradientelution with the IEC column may be favorable because of equivalentresolution as polyacrylamide gel electrophoresis (PAGE) and increasedloading capability when compared to SEC. In liquid affinitychromatography (LAC) interaction may be based on binding of the proteindue to mimicry of substrate, receptor, etc. The protein may be eluted byintroducing a competitive binding agent or altering the proteinconfiguration which may facilitate dissociation. HPLC may be coupledwith MS.

D. Electrophoresis.

Electrophoresis can be used for the analysis of the hemoglobin of thepresent invention. Electrophoresis can be gel electrophoresis orcapillary electrophoresis.

Gel Electrophoresis:

Gel electrophoresis is a technique that can be used for the separationof proteins. Separation of large (macro) molecules may depend upon twoforces: charge and mass. During electrophoresis, macromolecules areforced to move through the pores when the electrical current is applied.Their rate of migration through the electric field depends on thestrength of the field, size and shape of the molecules, relativehydrophobicity of the samples, and on the ionic strength and temperatureof the buffer in which the molecules are moving. Using this technologyit is possible to separate and identify protein molecules that differ byas little as a single amino acid. Also, gel electrophoresis allowsdetermination of crucial properties of a protein such as its isoelectricpoint and approximate molecular weight. Electrofocusing or isoelectricfocusing is a technique for separating different molecules by theirelectric charge differences, taking advantage of the fact that amolecule's charge changes as the pH of its surroundings changes.

Capillary Electrophoresis:

Capillary electrophoresis is a collection of a range of separationtechniques which may involve the application of high voltages acrossbuffer filled capillaries to achieve separations. The variations includeseparation based on size and charge differences between analytes (termedcapillary zone electrophoresis (CZE) or free solution CE (FSCE)),separation of neutral compounds using surfactant micelles (micellarelectrokinetic capillary chromatography (MECC) or sometimes referred toas MEKC) sieving of solutes through a gel network (capillary gelelectrophoresis, GCE), separation of cations (or anions) based onelectrophoretic mobility (capillary isotachophoresis, CITP), andseparation of zwitterionic solutes within a pH gradient (capillaryisoelectric focusing, CIEF). Capillary electrochromatography (CEC) canbe an associated electrokinetic separation technique which involvesapplying voltages across capillaries filled with silica gel stationaryphases. Separation selectivity in CEC can be a combination of bothelectrophoretic and chromatographic processes. Many of the CE separationtechniques rely on the presence of an electrically induced flow ofsolution (electroosmotic flow, EOF) within the capillary to pump solutestowards the detector. GCE and CIEF are of importance for the separationof biomolecules such as proteins.

E. Nuclear Magnetic Resonance (NMR).

NMR can be used for the analysis of the hemoglobin of the presentinvention. NMR spectroscopy is capable of determining the structures ofhemoglobin at atomic resolution. In addition, it is possible to studytime dependent phenomena with NMR, such as intramolecular dynamics inmacromolecules, reaction kinetics, molecular recognition or proteinfolding. Heteronuclei like ¹⁵N, ¹³C and ²H, can be incorporated inproteins by uniformly or selective isotopic labeling. Spectra from thesesamples can be drastically simplified. Additionally, some newinformation about structure and dynamics of macromolecules can bedetermined with these methods.

F. X-Ray Crystallography.

X-ray crystallography can be used for the analysis of the hemoglobin ofthe present invention. X-ray crystallography is a technique in which thepattern produced by the diffraction of X-rays through the closely spacedlattice of atoms in a crystal is recorded and then analyzed to revealthe nature of that lattice. This generally leads to an understanding ofthe material and molecular structure of a substance. The spacings in thecrystal lattice can be determined by using Bragg's law. The electronsthat surround the atoms, rather than the atomic nuclei themselves, arethe entities which physically interact with the incoming X-ray photons.This technique can be used to determine the structure of the hemoglobinof the present invention. X-ray diffraction is commonly carried outusing single crystals of a material, but if these are not available,microcrystalline powdered samples may also be used which may requiredifferent equipment.

G. Arrays.

Arrays can be used for the analysis of the hemoglobin of the presentinvention. Arrays involve performing parallel analysis of multiplesamples against known protein targets. The development of variousmicroarray platforms can enable and accelerate the determination ofprotein abundance, localization, and interactions in a cell or tissue.Microarrays provide a platform that allows identification of proteininteraction or function against a characterized set of proteins,antibodies, or peptides. Protein-based chips array proteins on a smallsurface and can directly measure the levels of proteins in tissues usingfluorescence-based imaging. Proteins can be arrayed on either flat solidphases or in capillary systems (microfluidic arrays), and severaldifferent proteins can be applied to these arrays. Nonspecific proteinstains can be then used to detect bound proteins.

H. Amino Acid Analysis.

In some embodiments, amino acid analysis (AAA) is a technique used inthe analysis of the hemoglobin of the present invention. AAA is aprocess to determine the quantities of each individual amino acid in aprotein. There can be four steps in amino acid analysis: hydrolysis,derivatization, separation of derivatized amino acids, and datainterpretation and calculations.

In the hydrolysis step, a known amount of internal standard (norleucine)may be added to the sample. The sample, containing at least 5 nmoles ofeach amino acid (i.e. 10 μg of protein) can be then transferred to ahydrolysis tube and dried under vacuum. The tube can be placed in a vialcontaining HCl and a small amount of phenol and the protein ishydrolyzed by the HCl vapors under vacuum. The hydrolysis is carried outfor about 24 h at about 110° C. Following hydrolysis, the sample can bedried.

Derivatization can be performed automatically on the amino acid analyzerby reacting the free amino acids, under basic conditions, for example,with phenylisothiocyanate (PITC) to produce phenylthiocarbamyl (PTC)amino acid derivatives. A standard solution containing a known amount(500 pmol) of 17 common free amino acids can also be loaded on aseparate amino acid analyzer sample spot and derivatized. This can beused to generate a calibration file that can be used to determine aminoacid content of the sample. Following derivatization, a methanolsolution containing the PTC-amino acids can be transferred to a narrowbore HPLC system using a reverse phase C18 silica column for separation.The buffer system used for separation can be for example, 50 mM sodiumacetate at pH 5.45 as buffer A and 70% acetonitrile/32 mM sodiumphosphate at pH 6.1 as buffer B. The program can be run using a gradientof buffer A and buffer B. Chromatographic peak areas can be identifiedand quantitated using a data analysis system that can be attached to theamino acid analyzer system.

Alternatively, the classical method of amino acid analysis of Moore andStein using ninhydrin may be used.

Clinical Chemistry Analysis Methods

A. Oxygen Transport.

A CO-oximeter is used for comprehensive hemoglobin analysis to establishsaturation, desaturation and methemoglobin levels. Ultravioletillumination is used to for oxygen transport tests including levels ofdeoxyhemoglobin (HHb), oxyhemoglobin (O₂Hb), methemoglobin (MetHb),carboxyhemoglobin (COHb), total hemoglobin (tHb), oxygen saturation(SO₂%), oxygen content (O₂Ct), and oxygen capacity (O₂Cap) in thehemoglobins of the invention. One suitable instrument is manufactured byNova Biomedical Instrumentation.

B. Electrolytes.

Electrolytes such as potassium, calcium, sodium chloride, and others aremeasured using standard electrolyte/chemistry analyzers. Suitableinstrumentation is produced by Nova Biomedical, Hitachi, Roche, amongothers.

C. Osmolality.

The osmolality of the hemoglobins of the invention is also measured.Freezing point depression is the methodology used to perform thisanalysis, in order to produce biocompatible volume expansion and oxygendelivery agents of the invention. Suitable instrumentation is availablefrom Advanced Instruments Inc., and can measure all osmoticially activesolutes within the range of 0.0 to 4000 mOsmol/kgH₂O.

D. Carbonic Anhydrase.

Carbonic anhydrase may be detected by a double sandwich ELISA, wherein apolystryene support is coated with rabbit anti-bovine CA, to which CA inthe samples will bind. The enzyme substrate reaction is quantified byvisible absorbance of the products of the reaction.

E. Phospholipid Level Reduction.

Phospholipid assays can be measured by HPLC and/or ELISA. The ELISAvalidation protocol is designed according to current USP guidelines fora Category II, quantitative assay to determine the presence ofphosphatidylcholines. The protocol includes validation oflinearity/range, accuracy and precision.

F. Assay for Endotoxin.

The final product, ready for infusion, must be endotoxin free. Endotoxinis actually material from bacterial cell walls, and is responsible forinitiation of a fever in the recipient, in low doses; while higherlevels will initiate a more serious constellation of symptoms. The LAL(Limulus Ameobocyte Lysate) kinetic-turbidometric assay was chosen overother assays, such as the chromogenic and the gel-clot, because of itsreproducible results and high degree of sensitivity.

The potency of Control Standard Endotoxin (CSE) used for routine testingis determined by comparison with Reference Standard Endotoxin (RSE),EC5, Lot F manufactured by the USPC. It is necessary to perform thiscomparison whenever an endotoxin other than the Referenced endotoxin isto be used for creating spikes and curves in routine testing. This is aconsequence of the fact that different lots of CSE have markedlydifferent potencies. The RSE/CSE comparison is performed by comparingone vial of RSE to four vials of the same lot of CSE and calculating anaverage potency. A standard curve is assayed in triplicate, with acoefficient of correlation of −0.98 or less required for qualification.Numerous CSE standard curves are run and one standard curve is archivedfor future testing. Inhibition/enhancement studies are performed on allproducts to be tested with the LAL assay. The LAL assay is performedusing the protocol of Associates of Cape Cod, Falmouth Technology Park,East Falmouth Mass. 02536-4445, using a Pyros Kinetix® Incubating TubeReader as manufactured by Associates of Cape Cod.

i. Reagent and Equipment Preparation

All reagents used for the kinetic-turbidometric assay are used accordingto the specific manufacturers' instructions with the following twoexceptions: 1) the LAL is reconstituted with 5 ml of Pyrosol™reconstitution buffer instead of LAL Reagent Water and 2) the CSE is notreconstituted with exactly 5 ml of LAL Reagent Water. The reconstitutionof LAL with buffer is performed to overcome extreme enhancement of theLAL assay by pure hemoglobin solutions. The CSE is reconstituted with anamount of water which will yield a final solution concentration of 1000EU/ml. The amount to be added is determined by standardizing the CSEagainst the USPC RSE and may be more or less than the 5 ml recommendedby Associates of Cape Cod. This yields a constant CSE solutionconcentration and prevents recalculation of endotoxin spikes every timethe CSE lot changes. All other reagents are used as directed.

All glassware is depyrogenated by heating to 180° C. for 4 h. Alldilutions and solution transfers are performed under a class 100 laminarflow hood. All pipette tips used are sterile and pyrogen-free.

ii. Determination of CSE Potency

One vial of RSE (10,000 EU/vial by definition) was reconstituted with 5ml of LAL Reagent Water to yield a 2,000 EU/ml solution. Two RSE curveswere run to cover full range of current Q.C. testing. The mid-rangecurve contained the following concentrations (EU/ml): 1.0, 0.5, 0.25,0.125, 0.0625, and 0.03125. The low range curve consisted of thefollowing concentrations (EU/ml): 0.004, 0.002, 0.001, 0.0005, 0.00025,and 0.0001. These curves were run in duplicate, linear regression wasperformed to determine the slope and Y-intercept of the curves, and thecurves were archived for the purpose of comparison with the CSE curves.

After the RSE curves had been run, 4 vials of CSE (500 ng/vial) werereconstituted with an amount of water which will yield a final solutionconcentration of 1000 EU/each. Dilutions of each vial were prepared ineach of the two ranges so that at least three concentrations of the CSEcurve would fall directly on the RSE curve. The mid-range curvecontained the following concentrations (ng/ml): 0.1, 0.05, 0.025,0.0125, 0.00625 and 0.003125. The low-range curve consisted of thefollowing concentrations (ng/ml): 0.004, 0.002, 0.001, 0.0005, 0.00025,and 0.0001. These curves were run in duplicate and the onset times wereinterpolated off the corresponding RSE curve.

The endotoxin concentration in EU/ml for each CSE standard was dividedby the corresponding concentration in ng/ml. Any onset times which didnot fall directly on the RSE curve, indicated on the raw data by anasterisk, were not included in the calculations. The resulting EU/ngpotencies for each standard were averaged to determine the CSE potencyin each range. The two range potencies were then averaged to determinethe potency of the CSE through the entire range of 1.0 to 0.001 EU/ml.The overall CSE potency was then used to calculate the amount of LALReagent Water to be added to each CSE Vial in the lot to yield a 1000EU/ml solution according to the following calculations:

${\frac{500\; \frac{ng}{vial}}{1000\; \frac{EU}{ml}} \times {potency}\; \frac{EU}{ng}} = {{water}\; \frac{ml}{vial}}$

iii. Test Method

100 μl of LAL is added to a depyrogenated 10×75 mm culture tubecontaining 400 μl of sample. The tube is vortexed gently forapproximately 2 seconds and placed in the incubation module of the LALdevice. Each tube is added individually in this manner. Timing isinitiated for each tube as the bottom of the tube actuates a mechanicalswitch. Tubes are incubated at 37.0±0.5° C. throughout the test. Noreadings are taken for the first 60 seconds; this allows time for thecontents of the tube to come to temperature and for air bubbles todisperse. For 60 to 120 seconds after tube insertion, photodetectors ineach well take 7 readings 10 seconds apart. The readings are thenaveraged and taken to represent 100% transmittance. This zeroing periodeliminates data errors due to tube imperfections and sample color orendogenous turbidity. Subsequent readings are taken every ten seconds,converted to transmittance, and then to optical density (OD). From 400to 550 seconds, the OD values collected are averaged and then subtractedfrom all subsequent OD values for the test. (This baseline correctioncompensates for the OD of the background.)

Onset time, T_(o), is defined as the number of seconds between placementof a sample in and incubating well and the development of an opticaldensity of 20 mAU. The endotoxin level is determined by comparing theonset times to an archived standard curve of log₁₀(T_(o)) versus log₁₀(known endotoxin concentration).

A small-programmed-computer is used to collect data from the LAL device.The LAL device software stores the OD values in data files and performsdata analysis upon command such as onset time correction, linearregression on standards, and endotoxin concentration determination.

All in-process samples are tested in duplicate, unspiked and spiked,with a four-lambda spike, where lambda equals the lowest standard on thestandard curve. In addition, a four-lambda spike in LAL Reagent Waterand unspiked LAL Reagent Water are tested. All final product samples aretested unspiked and spiked, in triplicate.

Results.

The levels of endotoxin in dXCMSFH are below 1 EU per/ml. In preferredembodiments of the process, the elimination of endotoxins to a levelbelow 0.01 EU are achieved and allow for complex usage and for largervolumes. This results in readings of 0.1 EU per ml to below 0.02 EU perml.

V. FORMULATIONS

1. Pharmaceutical Compositions Including Excipients, Routes ofAdministration and Dosages.

The deoxygenated stable NO blocked tetrameric Hb, and dXCMSFH, inparticular, of the present invention may be incorporated in conventionalpharmaceutical formulations (e.g. injectable solutions) for use intreating mammals in need thereof. Pharmaceutical compositions can beadministered by subcutaneous, intravenous, or intramuscular injection,or as large volume parenteral solutions and the like. In someembodiments, dXCMSFH of the present invention may be formulated byencapsulating Hb within liposomes. Liposome encapsulation is often usedin drug delivery to reduce the toxicity of encapsulated therapeuticagents, as well as to increase drug half-life. The liposome-encapsulatedhemoglobin (LEHb) encases Hb in a structure physiologically similar toRBCs, thus preventing Hb dissociation and its rapid clearance in theblood stream. The half-life of LEHb dispersions is dependent on thesurface chemistry of the bilayer, as well as the bilayer surface chargeand the vesicle size distribution. Hence, decreasing vesicle size andmodifying the vesicle surface can significantly increase the circulatorylifetime. Surface conjugation of liposomes with polyethylene-glycol(PEG) can extend the half-life. The uptake of liposomes by thereticuloendothelial system (RES) affects the LEHb concentration that canbe safely administered, since overloading the RES would impair theimmune system.

The deoxygenated stable NO-blocked tetrameric Hb and, in particular,dXCMSFH of the present invention can also be formulated into otherartificial blood and oxygen delivery therapeutic formulations. Suchformulations can include other components in addition to the dXCMSFH.For example, a parenteral therapeutic composition can comprise a sterileisotonic saline solution. The formulations can be either in a formsuitable for direct administration, or in a concentrated form requiringdilution prior to administration. The formulations of the presentinvention can thus contain between 0.001% and 90% (w/v) dXCMSFH. In someembodiments of the present invention, the extracellular hemoglobinsolution of dXCMSFH of the present invention may contain from about 5percent to about 20 percent, from about 5 percent to about 17 percent,from about 8 to about 14 percent, and about 10 percent hemoglobin insolution (% weight per volume). In some embodiments of the invention,the extracellular hemoglobin solution of dCMSFH may contain from about5% to about 7% hemoglobin in solution (% w/v). In some embodiments ofthe invention the solution containing dCMSFH contains about 6.4%hemoglobin. The selection of percent hemoglobin depends on the oncoticproperties of the chosen hemoglobin product. The hemoglobin solutionsformulated for use in the present invention may be normo-oncontic tohyperoncotic. The percent hemoglobin may be adjusted to obtain thedesired oncotic pressure for each indication.

dXCMSFH of the present invention can be used in compositions useful asblood substitutes and oxygen delivery therapeutics in any mammal thatuses red blood cells for oxygen transport. The mammals include but arenot limited to, human, livestock such as cattle, cat, horse, dog, sheep,goat, pig etc. In some embodiments of the invention, the mammal ishuman.

A dose of the dXCMSFH of the present invention can be from about 1 toabout 15,000 milligrams of hemoglobin per kilogram of patient bodyweight over the appropriate time period either from initial dose orrepeat dose. When used as an oxygen delivery composition, or as a bloodvolume supplement, the dosage may range between 100 to 7500 mg/kgpatient body weight, 500 to 5000 mg/kg body weight, or 700 to 3000 mg/kgbody weight. Thus, a dose for a human patient might be from a gram toover 1000 grams. It will be appreciated that the unit content of activeingredients contained in an individual dose of each dosage form need notin itself constitute an effective amount, as the necessary effectiveamount could be reached by administration of a number of individualdoses. The selection of dosage depends upon the dosage form utilized,the condition being treated, and the particular purpose to be achievedaccording to the determination of those skilled in the art.

For use in the present invention, the deoxygenated stable NO blockedtetrameric HB and, in particular, the dXCMSFH of the present inventioncan be dialyzed or exchanged by ultrafiltration into a physiologicallyacceptable solution. The dXCMSFH of the present invention may beformulated at a concentration of 50-150 g/l. The solution may comprise aphysiologically compatible electrolyte vehicle isosmotic with wholeblood and which may maintain the reversible oxygen-carrying and deliveryproperties of the hemoglobin. The physiologically acceptable solutioncan be, for example, physiological saline, a saline-glucose mixture,Ringer's acetate, Ringer's solution, lactated Ringer's solution,Locke-Ringer's solution, Krebs-Ringer's solution, Hartmann's balancedsaline, heparinized sodium citrate-citric acid-dextrose solution, andpolymeric plasma substitutes, such as polyethylene oxide, polyvinylpyrrolidone, polyvinyl alcohol and ethylene oxide-propylene glycolcondensates.

Each formulation according to the present invention may additionallycomprise inert constituents including pharmaceutically-acceptablecarriers, diluents, fillers, salts, and other materials well-known inthe art, the selection of which depends on the dosage form utilized, thecondition being treated, the particular purpose to be achieved accordingto the determination of the ordinarily skilled artisan in the field andthe properties of such additives. For example, the hemoglobin solutionof the present invention in addition to dXCMSFH may include 0-200 mM ofone or more physiological buffers, 0-200 mM of one or morecarbohydrates, 0-200 mM of one or more alcohols or poly alcohols, 0-200mM of one or more physiologically acceptable salts, and 0-1% of one ormore surfactants, 0-20 mM of a reducing agent. The hemoglobin solutionof the present invention in addition to dXCMSFH may include, 0-50 mMsodium gluconate, 0-50 mM of one or more carbohydrates (e.g. glucose,mannitol, sorbitol or others known to the art), 0-300 mM of one or morechloride salts and, optionally, 0-0.5% surfactant, e.g. Tween™[polysorbate 80], and/or 0-20 mM N-acetyl cysteine.

Administration of the dXCMSFH of the present invention can occur for aperiod of seconds to hours depending on the purpose of the hemoglobinusage. For example, when used as an oxygen carrier for the treatment ofsevere hemorrhage, the usual time course of administration is as rapidlyas possible. Typical infusion rates for hemoglobin solutions as volumeenhancer or oxygen therapeutics can be, for example, from about 100 ml/hto about 3000 ml/h, from about 1 ml/kg/h to about 300 ml/kg/h, or fromabout 1 ml/kg/h to about 25 ml/kg/h. In some embodiments of theinvention, the rates of administration may be higher.

Suitable compositions can also include 0-200 mM of one or more buffers(for example, acetate, phosphate, citrate, bicarbonate, or Goode'sbuffer). Salts such as sodium chloride, potassium chloride, sodiumacetate, calcium chloride, magnesium chloride can also be included inthe compositions of the invention. The salt can be in concentrations of0-2M.

In addition, the compositions of the invention can include one or morecarbohydrate (for example, reducing carbohydrates such as glucose,maltose, lactose or non-reducing carbohydrates such as sucrose,trehalose, raffinose, mannitol, isosucrose or stachyose) and one or morealcohol or poly alcohol (such as polyethylene glycols, propyleneglycols, dextrans, or polyols). The concentration of carbohydrate oralcohol can be 0-2 M.

The dXCMSFH of the present invention can also contain one or moresurfactant and 0-200 mM of one or more chelating agent (for example,ethylenediamine tetraacetic acid (EDTA), ethyleneglycol-bis(beta-aminoethyl ether) N,N,N,N′-tetraacetic acid (EGTA),ophenanthroline, diethylamine triamine pentaacetic acid (DTPA also knownas pentaacetic acid) and the like). The surfactant can be 0.005-1% ofthe composition. The compositions of the invention can be at pH of about6.0-9.5. In some embodiments, the composition may contain 0-150 mM NaCl,0-10 mM sodium phosphate, 0.01-0.1% surfactant, and/or 0-50 μM of one ormore chelating agents at pH 6.0-9.5. The formulation may contain 5 mMsodium phosphate, 150 mM NaCl, 0.025% to 0.08% polysorbate 80, and/or 25μM EDTA at pH 6.0-9.5.

Additional additives to the formulation can include anti-bacterialagent, oncotic pressure agent (e.g. albumin or polyethylene glycols) andother formulation acceptable salt, sugar and other excipients known inthe art. Each formulation according to the present invention canadditionally comprise constituents including carriers, diluents,fillers, salts, and other materials well-known in the art, the selectionof which depends upon the particular purpose to be achieved and theproperties of such additives which can be readily determined by oneskilled in the art. The compositions of the present invention can beformulated by any method known in the art. Such formulation methodsinclude, for example, simple mixing, sequential addition,emulsification, diafiltration and the like.

2. Packaging and Storage of the NO-Blocked Tetrameric Hb of theInvention, Including Both Stable (Cross Linked) and Unstabilized(Uncross Linked) Hb.

Various embodiments of the NO-blocked tetrameric Hb of the invention,including dXCMSFH, dCMSFH, and dTBSFH may be stored in conventional, andpreferably oxygen impermeable containers (for example, stainless steeltanks, glass containers, oxygen impermeable plastic bags, or plasticbags overwrapped with low oxygen permeable plastic bags wherein anoxygen scavenger is placed between the internal plastic bag and theoverwrapped plastic bag). In some preferred embodiments, the dXCMSFH,dCMSFH, or dTBSFH of the present invention is stored in the absence ofoxygen. The dXCMSFH, dCMSFH, or dTBSFH may be oxygenated prior to usesuch as, by way of example only, oxygenating before using in thecatheter for cardiac therapy. In some embodiments, the dXCMSFH, dCMSFH,or dTBSFH can be stored in oxygen permeable or oxygen impermeable(“anoxic”) containers in an oxygen controlled environment. Such oxygencontrolled environments can include, for example, glove boxes, glovebags, incubators and the like. Preferably the oxygen content of theoxygen controlled environment is low relative to atmospheric oxygenconcentrations (see, Kandler, R. L. et al., U.S. Pat. No. 5,352,773;herein incorporated by reference). In some embodiments of the presentinvention, the dXCMSFH, dCMSFH, or dTBSFH can be packaged in sealedTyvek or Mylar (polyethylene terephthalate) bags or pouches. In someembodiments, the dXCMSFH, dCMSFH, or dTBSFH of the present invention canbe lyophilized and stored as a powder. The preparations may be stored atroom or elevated temperature (Kandler et al., PCT Publication No. WO92/02239; Nho, PCT Publication No. WO 92/08478, both herein incorporatedby reference), or more preferably under refrigeration. In someembodiments, the dCMSFH or dTBSFH may be stored in HyClone BioProcessContainers™ for ease of shipping and further handling.

Where the package is an oxygen impermeable film, the container can bemanufactured from a variety of materials, including polymer films,(e.g., an essentially oxygen-impermeable polyester, ethylene vinylalcohol (EVOH), or nylon), and laminates thereof. Where the container isan oxygen impermeable overwrap, the container can be manufactured from avariety of materials, including polymer films, (e.g., an essentiallyoxygen-impermeable polyester, ethylene vinyl alcohol (EVOH), or nylon)and laminates, such as a transparent laminate (e.g. a silicon oxide orEVOH containing-laminate) or a metal foil laminate (e.g., a silver oraluminum foil laminate). The polymer can be a variety of polymericmaterials including, for example, a polyester layer (e.g., a 48 gaugepolyester), nylon or a polyolefin layer, such as polyethylene, ethylenevinyl acetate, or polypropylene or copolymers thereof.

The containers can be of a variety of constructions, including vials,cylinders, boxes, etc. In a preferred embodiment, the container is inthe form of a bag. A suitable bag can be formed by continuously bondingone or more (e.g., two) sheets at the perimeter(s) thereof to form atightly closed, oxygen impermeable, construction having a finablecenter. In the case of laminates comprising polyolefins, such as linearlow density, low density, medium or high density polyethylene orpolypropylene and copolymers thereof, the perimeter of the bag may bebonded or sealed using heat. It is well within the skill of the art todetermine the shape of the bag and the appropriate temperature togenerate a tightly closed, oxygen and/or moisture impermeableconstruction. Where the container is a film, such as a polyester film,the film can be rendered essentially oxygen-impermeable by a variety ofsuitable methods. The film can be laminated or otherwise treated toreduce or eliminate the oxygen permeability.

In some embodiments, one or more antioxidants, such as ascorbate(Wiesehahn, G. P. et al., U.S. Pat. No. 4,727,027; and, Kerwin, B. D. etal., U.S. Pat. No. 5,929,031), glutathione, N-acetylcysteine,methionine, tocopherol, butyl hydroxy toluene, butyl hydroxy anisole, orphenolic compounds (Osterber et al., PCT Publication No. WO 94/26286;and, Kerwin, B. D. et al., U.S. Pat. No. 5,929,031) may be added tofurther stabilize the dXCMSFH, dCMSFH, and dTBSFH (all references hereinincorporated by reference). Alternatively, and more preferably, thedXCMSFH of the present invention can be lyophilized and stored as apowder, or can be packaged in sealed Tyvek, or Mylar (polyethyleneterephthalate) bags or pouches. Packaging such as, Kerwin, B. D. et al.,U.S. Pat. No. 5,929,031, is herein incorporated by reference. In someembodiments, the dXCMSFH, dCMSFH, and dTBSFH in such storage containersmay be subjected to irradiation or other sterilization treatmentsufficient to extend the shelf-life of the compositions. An oxygenscavenger such as n-acetyl-cysteine may be included in the formulation.

The dXCMSFH, dCMSFH, and TBSFH of the present invention may be stored atsuitable storage temperatures for periods of two years or more, andpreferably for periods of two years or more, when stored in a low oxygenenvironment. Suitable storage temperatures for storage of one year ormore are between about 0° C. and about 40° C. The preferred storagetemperature range is between about 0° C. and about 25° C. The process ofmaking dXCMSFH, dCMSFH, and dTBSFH of the present invention includesmaintaining the steps of the process under conditions sufficient tominimize microbial growth, or bioburden, such as maintaining temperatureat less than about 20° C. and above 0° C.

VI. METHODS OF USE

The deoxygenated stable NO-blocked tetrameric Hb of the presentinvention, and, in particular, the dXCMSFH may be used to formpharmaceutical compositions that may be administered to recipients, forexample, by infusion, by intravenous or intra-arterial injection, or byother means. The dXCMSFH formulations of the present invention can beused in compositions useful as blood substitutes, volume expanderswithin the blood volume, and oxygen perfusion agents in any applicationwhere red blood cells are used. One application uses compositions of thepresent invention for the treatment of hemorrhage where blood volume islost and both fluid volume and oxygen delivery capacity must bereplaced. Moreover, because the deoxygenated stable NO-blockedtetrameric Hb of the present invention, and, in particular, dXCMSFH, canbe made pharmaceutically acceptable, the formulations of the presentinvention can not only deliver oxygen but also act as simple volumeexpanders that provide oncotic pressure due to the presence of the largehemoglobin protein molecule. The deoxygenated stable NO-blockedtetrameric Hb of the present invention, and, in particular, dXCMSFH, canthus be used as replacement for blood that is removed during surgicalprocedures where the patient's blood is removed and saved for reinfusionat the end of surgery or during recovery (e.g., acute normovolemichemodilution or hemoaugmentation, etc.).

A typical dose of the deoxygenated stable NO-blocked tetrameric Hb ofthe present invention, and, in particular, dXCMSFH as a blood substituteis from 10 mg to 7 grams or more of extracellular hemoglobin perkilogram of patient body weight. Thus, a typical dose for a humanpatient might be from a few grams to over 350 grams. It will beappreciated that the unit content of active ingredients contained in anindividual dose of each dosage form need not in itself constitute aneffective amount since the necessary effective amount could be reachedby administration of a plurality of administrations as injections, etc.The selection of dosage depends upon the dosage form utilized, thecondition being treated, and the particular purpose to be achievedaccording to the determination of the ordinarily skilled artisan in thefield.

In some embodiments of the invention, a solution of a deoxygenatedstable NO-blocked tetrameric Hb, for example, dXCMSFH, will containabout 5% to about 25% dXCMSFH by weight for administration to a mammal.In some preferred embodiments of the invention a solution of dXCMSFHwill contain about 7% to about 15% dXCMSFH by weight for administrationto a mammal. In some other preferred embodiments of the invention, asolution of dXCMSFH will be 10% by weight of dXCMSFH for administrationto a mammal. In some embodiments of the invention, a dose to beadministered to a mammal contains about 7 g of dXCMSFH. In someembodiments of the invention a dose to be administered to a mammalcontains about 1 g of a deoxygenated stable NO-blocked tetrameric Hb,and in particular, dXCMSFH. In some embodiments of the invention, anexemplary unit of production for use in a therapeutic setting is acontainer with 500 ml of a 0.5 mmol solution of dXCMSFH (about 64 g/L,or about 6.4% by weight in solution). The larger unit solutions may beused for replacement of blood or for augmenting oxygen delivery for anumber of therapeutic interventions. The smaller unit solutions may beused for labeling and diagnostic purposes, as well as therapeuticinterventions. The smaller unit solutions may, in a preferredembodiment, contain a solution of dXCMSFH of up to 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21, 22%, 23%,24%, or up to 25% by weight of dXCMSFH. In another embodiment, asolution may contain dXCMSFH with a concentration as low as 2%, 3%, 4%,5%, 6%, 7%, 8% 9%, 10%, 11%, 12%, 13% or 14%, by weight of dXCMSFH.

Administration of the deoxygenated stable NO-blocked tetrameric Hb ofthe present invention, and, in particular, dXCMSFH, can occur for aperiod of seconds to hours depending on the purpose of the hemoglobinusage. For example, as a volume augmentation therapy, the usual timecourse of administration is as rapid as possible. Typical infusion ratesfor hemoglobin solutions as oxygen delivery/perfusion agents or volumeenhancers can be from about 100 ml to 3000 ml/h. However, when used tostimulate hematopoiesis, administration can be made more slowly andtherefore administration rates can be slower because the dosage of thedeoxygenated stable NO-blocked tetrameric Hb of the present invention,and, in particular, dXCMSFH may be much less than dosages that may berequired to treat hemorrhage.

In some embodiments, the deoxygenated stable NO-blocked tetrameric Hb ofthe present invention, and, in particular, dXCMSFH, can be used to treatanemia as caused by renal failure, diabetes, AIDS, chemotherapy,radiation therapy, hepatitis, G.I. blood loss, iron deficiency,menorrhagia, and the like, by providing additional oxygen deliverycapacity in a mammal that is suffering from anemia, as well as bystimulating hematopoiesis, providing an effective iron supplement tosupport RBC production, and by serving as an adjuvant to erythropoietintherapy.

Likewise, the deoxygenated stable NO-blocked tetrameric Hb of thepresent invention, and, in particular, dXCMSFH, can be used to provideadditional oxygen delivery capacity to a mammal (such as an athlete,soldier, mountaineer, aviator, smoke victim, etc.) desiring suchadditional oxygen delivery capacity. Such additional oxygen deliverycapacity can be used to overcome environmental (i.e, for example, highaltitudes and smoke inhalation) and physical (i.e., for example, acuteperformance demands) stresses. The stable NO-blocked tetrameric Hbs ofthe present invention, and in particular, dXCMSFH, thus are useful intreating carbon monoxide poisoning and its concurrent hypoxia andischemia, as the compounds and compositions of the present invention cansupply oxygen to tissue while the carbon monoxide bound cellularhemoglobin is being eliminated, thus bridging the oxygen needs of thepatient until new RBCs are produced.

The deoxygenated stable NO-blocked tetrameric Hbs of the presentinvention, and, in particular, dXCMSFH, can be used for applicationsrequiring administration to a mammal of high volumes of hemoglobin aswell as in situations where only a small volume of the hemoglobin of thepresent invention is administered. The deoxygenated stable NO-blockedtetrameric Hb of the present invention, and, in particular, dXCMSFH canbe used in applications during surgery where large volumes of blood arenormally lost, or in treatment of trauma victims who have lost largevolumes of blood. This can include both civilian accidents and militarysituations.

The deoxygenated stable NO-blocked tetrameric Hb of the presentinvention, and, in particular, dXCMSFH may be used as a blood substitutein veterinary clinical applications.

In addition, because the distribution throughout the vasculature of thedeoxygenated stable NO-blocked tetrameric Hb of the present invention,and, in particular, dXCMSFH, is not limited by viscosity or by the sizeof red blood cells, the compositions of the present invention can beused to deliver oxygen to areas that red blood cells cannot penetrate.These areas can include any tissue areas that are located downstream ofobstructions to red blood cell flow, such as areas downstream ofthrombi, sickle cell occlusions, arterial occlusions, angioplastyballoons, surgical instrumentation, tissues that are suffering fromoxygen starvation or are hypoxic, and the like.

Additionally, all types of tissue ischemia, including ischemic events inthe brain, can be treated using the methods of the instant invention.Such tissue ischemias include, for example, stroke, emerging stroke,transient ischemic attacks, myocardial stunning and hibernation, acuteor unstable angina, emerging angina, infarct, and the like. The recoveryof tissues from physical damage such as burns can also be accelerated bypretreatment with the hemoglobin of the present invention, which allowsincreased perfusion and oxygenation of the tissues which may also reduceinfection risk. The use of the stable NO-blocked tetrameric Hbs of thepresent invention also will allow for better oxygen uptake in the lungsdue to better distribution of these smaller molecules within smallcapillaries. In and after cosmetic surgery, fine tissue beds suffermicrocirculatory disruption, thereby losing flow of RBCs. Use of thestable NO-blocked tetrameric Hbs of the present invention provide betteroxygenation for tissue metabolism and regrowth, to decrease scarringwith its loss of vascularization.

The deoxygenated stable NO-blocked tetrameric Hb of the presentinvention, and, in particular, dXCMSFH, can be used for the treatment ofsickle cell anemia patients. Sickle cell anemia patients invasoocclusive crisis are currently treated by transfusion of red bloodcells in conjunction with dilution and pain management. The deoxygenatedstable NO-blocked tetrameric Hb of the present invention, and, inparticular, dXCMSFH, may not only deliver oxygen thereby preventingfurther sickling (as do red blood cells), they may also penetratevessels already occluded with deformed red cells to better alleviatepain and minimize tissue damage. Also, frequent transfusions in thesickle cell anemia population may result in alloimmunization to redcells and to platelets, an adverse effect that would be avoided by useof hemoglobin of the present invention. The deoxygenated stableNO-blocked tetrameric Hb of the present invention, and, in particular,dXCMSFH, n offer a significant therapeutic advantage in treatment ofsickle cell anemia patients, since they elicit a lesser degree ofvasoconstriction or none at all. This is an advantage in the treatmentof vasoocclusive crisis, and is also an advantage in other treatments ofsickle cell anemia patients in situations where there is a risk ofsudden onset of vasoocclusive crisis. For example, dXCMSFH of thepresent invention may be used in place of packed red cells forpreoperative transfusion of sickle cell anemia patients to minimize riskof anesthesia. The deoxygenated stable NO-blocked tetrameric Hb of thepresent invention, and, in particular, dXCMSFH may also be administeredperiodically to minimize risk of stroke.

The stable NO-blocked Hb of the present invention, i.e, re-oxygenatedXCMSFH can perfuse because of its size, and deliver oxygen to tissuebeds that would normally be dependent upon diffusion alone due to thepoor perfusion of bulky large red blood cells. For example, thecompounds and compositions of the present invention can be used as atissue protectant in acute coronary syndrome (ACS) and intransplantation, where the area of insult or harvested organ is perfusedduring stopped flow situations. This may prevent reperfusion injury andallow for the salvage and preservation of tissues that have beenperfused, with subsequent normal circulation. Additionally, intransplantation procedures, organs may be prepared for harvesting byflushing with the compounds and compositions of the present invention toremove native blood agents and components prior to removal and continueto support tissue viability as discussed above.

The compounds and compositions of the present invention may also beutilized as a wound healing reagent where the molecular size and oxygendelivery capabilities may yield superior perfusion in poorlyvascularized regions such as, for example, diabetic foot injuries,recovery from cardiac revascularization and post surgical recovery, i.e.for example, cosmetic surgery or cancer resection breast reconstruction,where RBCs may not perfuse well due to size or rigidity. Anotherapplication of the stable NO-blocked tetrameric Hbs of the presentinvention may be in poorly vascularized tumor tissue beds of cancercells where appropriate use of the invention can allow for an increasein oxygen tension and allow for more effective use of radiation therapyand for the enhancement of oxygen dependent pharmaceutical agents.

The deoxygenated stable NO-blocked tetrameric Hb of the presentinvention, and, in particular, dXCMSFH, may be used as a small moleculeinhibitor of nitric oxide for cardiogenic shock. Cardiogenic shockafflicts a significant number of patients presenting with acutemyocardial infarction, whereby circulatory shutdown occurs after theinfarct. Despite intervention with catheters or bypassgrafting, themortality rate is about 50%. The use of the compounds and compositionsof the present invention may provide the level of oxygenation to heartand blood vessels to forestall excessive production of nitric oxide andsupport survival past the critical initial thirty day post infarct timeperiod. Survival is greatly enhanced after this timepoint.

The deoxygenated stable NO-blocked tetrameric Hb of the presentinvention, and, in particular, dXCMSFH contains iron, and as such, maybe detected via MRI (magnetic resonance imaging). Thus, in someembodiments, the present invention contemplates the use of deoxygenatedstable NO-blocked tetrameric Hb of the present invention, and, inparticular, dXCMSFH as an imaging agent.

The present invention also concerns implantable delivery devices (suchas cartridges, implants, etc.) that contain deoxygenated stableNO-blocked tetrameric Hb of the present invention, and, in particular,dXCMSFH, and that are capable of releasing dXCMSFH, for example, intothe circulation in response to a sensed need for increased oxygendelivery capacity. In some embodiments, such devices can deliverdXCMSFH, for example, at a constant rate, so as to facilitateerythropoiesis (either alone, or in combination with erythropoietin). Insome embodiments, the devices can be controlled by sensing means (suchas electronic probes of hemoglobin, O₂ level, CO₂ level, etc.) so as todeliver the deoxygenated stable NO-blocked tetrameric Hb of the presentinvention at a rate commensurate with the patient's oxygen deliverycapacity needs. Such sensing means may themselves be implantable, orpart of the implanted device, or may be located extracorporeally. Insome embodiments, such devices may be used to accomplish or facilitatethe hemo-diagnosis of individuals.

The deoxygenated stable NO-blocked tetrameric Hb of the presentinvention, and, in particular, dXCMSFH may also be used to formnon-pharmaceutical compositions that can be used, for example, asreference standards for analytical instrumentation needing suchreference standards, reagent solutions, control of gas content of cellcultures; for example by in vitro delivery of oxygen to a cell culture,and removal of oxygen from solutions. Additionally, the dXCMSFH of thepresent invention may be used to oxygenate donated tissues and organsduring transport.

The deoxygenated stable NO-blocked tetrameric Hb of the presentinvention, and, in particular, dXCMSFH may be used to scavenge endotoxinfrom surfaces or liquids. The invention thus contemplates devices, suchas cartridges, filters, beads, columns, tubing, and the like thatcontain the deoxygenated stable NO-blocked tetrameric Hb of the presentinvention. Liquids, such as water, saline, culture medium, albuminsolutions, etc., may be treated by passage over or through such devicesin order to remove endotoxin that may be present in such liquids, or tolessen the concentration of endotoxin present in such liquids. Thedeoxygenated stable NO-blocked tetrameric Hb of such devices ispreferably immobilized (as by affinity, ionic, or covalent bonding,etc.) to solid supports present in such devices. In some embodiments,the deoxygenated stable NO-blocked tetrameric Hb is bound to beads thatmay be added to the liquids being treated, and then subsequently removed(as by filtration, or affinity immobilization). In some embodiments, thebeads may be of ferromagnetic or paramagnetic metal, or may bethemselves magnetic, such that they may be readily separated from thetreated liquid by magnetic means.

The deoxygenated thiol blocked Hb, both cross linked and uncross linked,i.e., dCMSFH, dTBSFH, and dXCMSFH, can be used to remove oxygen fromsolutions requiring the removal of oxygen, and as reference standardsfor analytical assays and instrumentation. The deoxygenated thiolblocked Hb, both cross linked and uncross linked, i.e., CMSFH, TBSFH,and XCMSFH, can also be used in vitro to enhance cell growth in cellculture by maintaining oxygen levels.

The re-oxygenated stable NO-blocked tetrameric Hb of the presentinvention, and in particular, XCMSFH, can be used for the use invisualizing intravascular space. Present optical techniques for theobservation of vascular walls are relegated to non-optical techniquesdue to the opaque effects of the transfusion of red blood cells. Thestable NO-blocked Hb of the present invention, i.e., XCMSFH may not onlydeliver oxygen thereby preventing ischemia, but they may also present anoptically translucent field of view to allow for visualization of tissuebeds in situ for the determination of pathology; cancer, vulnerableplaques, lipid damage, stent placement, etc, using visible light in thered wavelength band, for example, to illuminate the targeted feature.The use of Optical Coherence Tomography may be expanded by employing thestable NO-blocked tetrameric Hbs of the present invention. Intermittentsaline flushes are currently employed to create transient visual fieldsin-vivo, but superior visualization and sustained examinations may bepossible with use of the present stable NO-blocked tetrameric Hb, whichcan continue to oxygenate the local area.

VII. EXAMPLES Example 1 Comparison of Two Methods of Initial Processingof Whole Blood

Materials: Bovine Blood Collection:

Bovine blood is collected in a 1 gallon collection container which mayhold 100 ml of 6% sodium ethylenediaminetetraacetic acid (EDTA) solutionand is cooled in ice.

The whole bovine arterial blood is divided into Batch A and Batch B.Batch A consists of 2200 ml of whole blood and is washed in thehaemonetics Cell Saver 5 to obtain concentrated red blood cells free ofplatelets, clotting factors, extra cellular potassium, anticoagulants,and cell stroma (Method A). Batch B consists of 1800 ml of whole bloodand is washed on a Millipore 0.65 μm filter (Method B).

Method A. Cell Saver 5 Removal of Plasma Proteins:

Cell Saver 5 from Haemonetics is used to concentrate erythrocytes fromother components in freshly collected anticoagulated bovine blood. Itmay be desirable not to fracture either leucocytes or erythrocytes atthis point.

After being passed through a coarse filter of 150 μm, the cells arewashed in a spinning bowl holding 225 ml of packed red blood cells andwashed with 3 liters of saline in a reverse flow from the outside edgeof the bowl towards the center. The centrifugation is gentle, and someof the WBCs are eluted in the wash, which may be desirable. Table 4shows the progress of serum protein removal at 500 ml increments. It isa continuous flow technique. Sample in Table 4 is the sample volumeapplied to the bowl. Progress is followed by reading the proteinconcentration at 280 nm spectrophotometrically, with the values given inA₂₈₀ units (absorbance units at 290 nm). The data points in Table 4 aretaken at the indicated points during the washing process. The resultsindicate that when a full bowl of red cells is washed with 3 liters ofNS, there is a greater than 3 log reduction in serum proteins. This alsoinfers a greater than 3 log reduction in viruses, prions, etc that arenot bound to red cell membranes. All values in Table 4 are corrected fordilution.

TABLE 4 Cell Saver 5 removal of plasma proteins Sample status A₂₈₀Absorbance Units A₂₈₀ of initial crude plasma containing sample 277.35A₂₈₀ of filtrate after 500 cc NS 43.20 A₂₈₀ of filtrate after 1000 cc NS7.69 A₂₈₀ of filtrate after 1500 cc NS 0.86 A₂₈₀ of filtrate after 2000cc NS 0.05

Method B. Millipore 0.65 μm Filter Removal of Plasma Proteins:

Batch B is passed through a 150 μm filter. The material is then filteredwith a tangential flow membrane that will pass plasma proteins butretain cellular components such as leucocytes and erythrocytes. Thetangential flow membrane filtration can be slower but it may requireless labor as it can run unattended. It can be more suitable forscaleup. Other types of large scale centrifuges may be used. The resultsof this continuous diafiltration are shown in Table 5, where all resultsare corrected for dilution. This method reveals a greater than 3 logreduction in plasma proteins, which also implies a similar log reductionin viruses, prions, etc.

TABLE 5 Millipore 0.65 μm filter removal of plasma proteins Samplestatus A₂₈₀ Absorbance Units A₂₈₀ of plasma in 1000 cc blood 221.6 A₂₈₀of filtrate after 1000 cc NS 82.1 A₂₈₀ of filtrate after 2000 cc NS 26.5A₂₈₀ of filtrate after 3000 cc NS 8.07 A₂₈₀ of filtrate after 4000 cc NS2.76 A₂₈₀ of filtrate after 5000 cc NS 0.81 A₂₈₀ of filtrate after 6000cc NS 0.29 A₂₈₀ of filtrate after 7000 cc NS 0.102

Evaluation of Leucocyte Loss/Removal.

It is desirable during the preparation of hemoglobin to remove any WBCsto remove granolocyte proteolytic enzymes from the hemoglobin solution.Thus at the stage of removing plasma proteins it is desirable to removethe WBCs without causing their lysis. The Cell Saver 5 technology canremove some of the WBCs in the floating buffy coat duringcentrifugation. However, the tangential flow membrane may retain all ofthe WBCs so an observed loss of WBCs may mean that cell lysis of theWBCs had occurred. Table 6 shows evaluation of the conservation of WBCsafter filtration with Cell Saver 5 or Millipore 0.65 μm filter. Whenappropriately corrected for volume, both methods provide adequateprotection from leucocyte lysis in the presence of RBCs.

TABLE 6 Evaluation of leucocyte loss/removal Sample Initial WBC FinalWBC (Vol Adj) % Recovery Cell Saver 5 5.79 × 10³ 6.10 × 10³ 100%Millipore 0.65 μm 5.79 × 10³ 5.63 × 10³ 100%

Batches A and B are then refrigerated for 8 h, whereupon both batchesare passed through a Baxter leuko-reducing filter, which also removesviral materials. Batch A yields 1500 ml of RBCs while Batch B yields1200 ml of RBCs. Samples are extracted from each batch throughout thecourse of cleaning.

Lysing Cells and Removing Stroma.

The 1500 ml of RBCs from Batch A are diluted with 6000 ml of DI water.After allowing the cells 45 seconds to lyse, 750 ml of 9% N saline (NS)is added to the solution to minimize the lysing of any leukocytespresent. The 1200 ml of RBCs from Batch B are lysed with 4800 ml of DIwater. No saline is added to Batch B at this point. Next, both batchesare passed over a 0.22 micron Pellicon filter. Once the hemoglobin hasbeen filtered out and collected in a separate flask, it is passed over asecond 10K Dalton Pellicon filter. This filters out any saline presentwhich is discarded. The pure hemoglobin is recirculated into theoriginal flask and is concentrated to a desired percentage, such as13.5% (w/v).

Blocking Sulfhydryls of Hemoglobin with Iodoacetamide (IAM).

After the samples are concentrated to 13.5% Hb, oxygen is removed aspreviously described, and the pH is adjusted to 7.4 with 0.1M sodiumphosphate buffer. Oxygen remaining is <10 ppb. Two molar equivalents ofIAM per mole of dSFH are added and the reaction is allowed to proceedfor 1 h. Progress of the reaction is monitored by an iodide electrode.Unreacted IAM by ultrafiltration using PBS. Once iodide is removed theintermediate dCMSFH is stable and may be is packaged in oxygen barriercontainers and can be safely stored at room temperature.

Deoxygenation and Cross-Linking:

The stable intermediate product, dCMSFH, is again placed in an oxygenfree (<10 ppm) environment and dissolved oxygen removed to a level ofless than 0.010 ppm. Membrane contactor technology can be the methodutilized to deoxygenate the hemoglobin in a controlled atmosphere withan oxygen level of less than 10 ppm. An initial oxygen saturationreading is taken for both batches using a polorgraphic dissolved oxygenprobe. Batch A has an initial reading at 4 mg/L. Batch B has an initialreading at 7 mg/L. The hemoglobin is pumped and recirculated through themembrane contactor using a peristaltic pump at a flow rate of 600 ml/minwith applied vacuum pressure of >28.5 in/Hg. The final oxygen saturationlevel for both batches is <0.01 ppm. The batches are then pH adjusted to8.4 with 0.5M NaOH for cross-linking. Once the pH is adjusted, 2.94 g ofbis-3,5-dibromosalicyl fumarate (DBSF) is added to Batch A (2 molarequivalents per sulfhydryl) while 1.47 g is added to Batch B (2 molarequivalents per sulfhydryl). Once cross-linking is complete, pH isadjusted with 0.5M citric acid back to 7.4 and the batches are stored inair-tight containers in the refrigerator.

The reaction is monitored by Capillary gel electrophoretic analysis isperformed using a Beckman CoulterPA-800® Proteomics instrument withstandard sample preparation, to determine the extent of cross-linking tomonitor the reaction time course. The reaction is complete when 95% ormore of the tetramer are cross-linked (data not shown). The samples arealso evaluated in a Hemox Analyzer for the recording of blood oxygenequilibrium curves based on dual wavelength spectrophotometry.

Comparison of Method A and Method B Overall:

The Method A and Method B purifications are run side by side to compareefficiency in releasing Hb while removing stroma and preventingleucocyte lysis. Either method will provide purified materials ofacceptable quality, and both methods will provide purified materials ofacceptable quality, and a combination of both methods may also beenvisioned to be used in the methods of the invention.

Example 2 Lysing White Blood Cells

Determination of the relative time of lysing of WBCs is demonstrated.Preferential lysing of RBCs relative to WBCs allows optimization of redblood cell lysis to obtain the maximum amount of hemoglobin, withoutalso introducing proteases from lysed WBCs.

Procedure:

2000 mls of whole blood is filtered through only the 100 g reservoirfilter of the Cell Saver 5. Seven beakers are then filled with 200 mlsof blood. One beaker is designated as the control. The other six beakersare then assigned a specific time for lysing at times of 30 seconds andthen 1, 2, 3, 4, and 5 minutes. The control beaker is started and 910mls of 0.9% saline solution is added. At time increments of 30 seconds,1, 2, 3, 4, and 5 minutes a 10 ml sample is taken for white blood cellanalysis.

For the remaining six beakers, 800 ml of DI water is added. After 30seconds 110 ml of 9% saline is added to the first beaker to stop lysing.After being allowed to stir for approximately 30 seconds, a 10 ml sampleis taken for white blood cell analysis. After 1 minute, 110 ml of 9%saline is then added to the second beaker. Again after being allowed tostir for 30 seconds, a 10 ml sample is taken. For the remaining fourbeakers, 110 ml of 9% saline is added at 2, 3, 4 and 5 minutes to stoplysing. After each time increment, a 10 ml sample is taken from each forwhite blood cell analysis via standard WBC quantitation.

Conclusion:

As seen in FIG. 2, the lysing of the white blood cells can take placebetween 2 and 3 minutes. So in order to optimize red blood cell lysis,lysing can be stopped at two minutes. Due to the addition of DI waterand saline, the volume is increased. The results shown in Table 7 arecorrected for dilution.

TABLE 7 Determination of Relative Lysing Time of WBCs ControlExperimental (K/mm³) (K/mm³) Time (Control Beaker) (Beakers 1-6) 30seconds 5.9 5.2  1 minute 5.4 4.9  2 minutes 5.8 5.0  3 minutes 7.4 4.5 4 minutes 6.8 4.0  5 minutes 5.7 3.4

Example 3 Rabbit Safety Trial

Materials:

Domestic rabbits are raised and treated by standard animal husbandry. IVaccess is established with a 22 or 24 gauge catheter into a shaventopically anesthetized ear vein for dXCMSFH infusion. IV infusion ismetered with a syringe pump; the total volume usually given over 45-60minutes. If blood is removed from rabbits, it is performed by insertinga 20-22 gauge catheter into the artery of the other ear. Procedures andinfusions are done using a Velcro cloth wrap type restraint.

Methods: Protocol A:

Rabbits are prepared for infusion as above dXCMSFH to be administeredhas a p50 for O₂ of 28-32 as determined on a TCS Hemox-Analyzer at 37°C. in pH 7.40 buffered NS, and is 12% w/v for the modified hemoglobin ofthe invention. The amount of dXCMSFH to be infused is based upon 10% ofthe estimated blood volume of a rabbit (56 ml/kg). This amount is placedinto the syringe, and infused by the pump over 45-60 minutes. The IVcatheter is then removed from the rabbit's ear and the rabbit returnedto its cage for observation. The control rabbits receive Hb without theNO blockage chemical modification.

Protocol B:

Venous and arterial access is prepared as above. A significant amount ofrabbit blood, 54-75 cc, is removed from the arterial catheter whilesimultaneously an identical volume of dXCMSFH is infused through thevenous access. This procedure is accomplished in 12-20 minutes totaltime. On subsequent days 2 and 3, 15 cc of dXCMSFH is infused throughthe heparin locked venous catheter over a 30 minute interval, twice eachday with 4 h between infusions.

Results:

Twenty-five (25) rabbits are infused according to protocol A, over anapproximately 1 h period as shown in Table 8. All of these rabbits livebeyond 72 h up to 75 days, without any deaths. Three (3) rabbits areinfused with Hb without the NO blockage chemical modification. All ofthese rabbits die within 7-12 minutes of starting the infusion, as shownin Table 9. Four (4) rabbits receive large amounts of dXCMSFH, accordingto protocol B, as shown in Table 10. All of these rabbits are alive andwell for more than two weeks, without any subsequent deaths. The rabbitstreated with dXCMSFH in either protocol A or B show 100% survival and noobvious signs of morbidity. Therefore, experimental subjects receiving650-7500 mg/kg of XCMSFH tolerate the experimental protocols A and Bwell, while the treatment group receiving cell free Hb, with no furthermodifications, at levels of 125-160 mg/kg, expire upon firstadministration.

TABLE 8 Dose Rabbit Weight dXCMSFH ID (Kg) (mg/Kg) Outcome 11 2.47 567survive 15 2.4 583 survive 13 2.62 534 survive B7 2.36 636 survive CO2.52 476 survive B3 2.24 670 survive B1 2.8 714 survive C02 2.65 566survive E03 2.38 630 survive 04 2.1 1143 survive 07 2.26 1062 survive C82.39 1004 survive MH2 3.12 808 survive LHD 2.78 647 survive D03 2.3 783survive D01 2.06 874 survive 05 2.29 629 survive 03 2.02 713 survive 012.12 679 survive JSD 2.13 676 survive D04 2.26 637 survive MH4 2.51 574survive K03 2.27 634 survive D08 2.28 632 survive D12 2.42 595 survive

TABLE 9 Dose Weight Elapsed dSFH Rabbit ID (Kg) time (min) (mg/Kg)Outcome 06 2.04 7 98 expired 10 2.39 12 126 expired 09 2.42 8 83 expired

TABLE 10 Dose dXCMSFH dXCMSFH Rabbit ID Weight (Kg) (mg) (mg/Kg) OutcomeB03 3.1 12000 3871 survive F02 2.79 11500 4122 survive F01 3.02 138004570 survive F05 2.65 12700 4792 survive

Example 4 Pie Safety Trial

Materials and Methods:

Twelve piglets weighing 10-16 kilogram were studied. Prior to inclusionin the study, noninvasive screening by 2D echocardiogram for cardiacanomalies and aortic valve diameter measurements were performed. 5 mg ofLasix 40 mg/ml concentration was administered to each piglet immediatelyafter establishing an IV, prior to initiating the top loading of theXCMSFH solution The XCMSFH solution had a p50 of 32 for Oxygen and aconcentration of 12 gram percent (120 mg XCMSFH/ml). Each pigletreceived 1200 mg XCMSFH/kg body weight.

Non invasive cardiac data was obtained using a blood pressure cuff on ahind limb. Doppler ultrasound (USCOM) was used to measure the beat tobeat cardiac output at various times throughout the infusion. The dataselected for analysis represented the best ultrasound wave form obtainedat any time point. Top loading the addition of study material waslimited by the upper boundary of fluids producing congestive heartfailure.

An amount equal to 14.3 percent of the calculated blood volume (70 mlblood/kg body weight) was infused through a peripheral IV over thecourse of one hour. Subject pigs were not anesthetized, sedated, orinvasively monitored.

Results:

FIGS. 7A-D represent, in order, cardiac output, systemic vascularresistance, systolic blood pressure and diastolic blood pressure as afunction of XCMSFH infused, corrected for body weight. All of thepiglets tolerated the infusion well; there were no subject deaths. Aleast squares method of correlating variables was used to evaluate thedata, providing slope and intercept. It is readily apparent that thereis great variability in the data of any parameter, regardless of theamount of XCMSFH infused. This relates to the fact that the subjectswere not sedated, nor restrained. Arousal, toileting, and handlingseemed to account for most of the variation, though there wassignificant variation between subjects' parameters even before theinitiation of any of the experiment.

The results of this study showed that the average (max, min) for theseindices were: Cardiac Output, 1.12 L/min (2.7, 0.45); Systemic VascularResistance, 7008 dynes·sec/cm⁵ (21590-3364); Systolic Blood Pressure,156.69 mmHg (193-130); and Diastolic Blood Pressure, 95.75 mmHg(113-72); respectively. The baseline values for Cardiac Output were 1.26L/min (1.62, 0.72); Systemic Vascular Resistance 9588 dynes·sec/cm⁵(12662-4991); Systolic Blood Pressure 140 mmHg (159-123); and DiastolicBlood Pressure, 84.25 mmHg (111-72); respectively. Cardiac output wasobserved to decrease slightly (<5%), while the systemic resistanceincreased by approximately 30%. Both systolic and diastolic bloodpressure increased slightly (12 & 14%, respectively) during theinfusion. The relationship between the varied dose overload of XCMSFHand the hemodynamic indices are represented in FIGS. 7A-D. Although, theeffect of XCMSFH on these indices is minimal, this effect seems to bedependent on the % volume overload delivered.

In this study, significant variability was found for all parametersapparently independent of the amount of XCMSFH infused. The amount ofminimal change in these cardiac parameters can be attributed to theincrease in blood volume, essentially fluid overloaded subjects. Resultsof this study indicate that all subjects survived the experiment, andthere were minimal changes in the cardiac parameters observed relatingto vasoactivity.

Prion Safety:

A number of measures are taken to ensure that the Hb of the inventionare prion free. Selection of suitable animals is an initial step,choosing only animals from a closed herd, which have been fed no animalprotein, given no antibiotics, and which are less than 30 months old. Asecond point for prevention of contamination is scrupulous attention toavoidance of mixing brain matter into blood. The sacrificial method ofthe “mushroom stunner” approach is chosen to eliminate the possibilityof brain matter contamination, and thus eliminate potential introductionof prion containing materials into the collected blood. Further, whenthe Hb is processed, the washing procedure to remove plasma proteinswill also remove prions. Additionally, when the Hb is filtered throughthe 300,00 Da molecular weight filter, any prions can be eliminated.Lastly, the Hb of the invention is processed through the Pall filter toremove leucocytes. At this point, small formed bodies such as prions andviruses can be removed. All of these precautions operate to secure thesafety of the Hb of the invention for use in human therapeutics andemergency procedures.

Deoxygenated and Oxygenated States.

The NO-blocked and stable NO-blocked tetrameric hemoglobins of theinvention are packaged for storage and transport as deoxygenatedspecies. For many therapeutic applications, the modified hemoglobins areused in the deoxygenated state. For applications where perfusion isrequired, for example, clearing a field of living tissue forobservation, perfusing an ischemic region, or maintaining an organex-vivo prior to transplantation, the modified hemoglobins may be usedin their re-oxygenated states to support tissue function.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A blood substitute suitable for administration to a human patient,the blood substitute comprising: a tetrameric hemoglobin; and apharmaceutically acceptable carrier; wherein said tetrameric hemoglobincomprises four bovine polypeptides covalently cross-linked together; andwherein said tetrameric hemoglobin includes at least one cysteine moietychemically modified with a thiol-protecting group such that said atleast one cysteine moiety is incapable of binding nitric oxide; andwherein said tetrameric hemoglobin has a p50 for oxygen greater thanthat of whole human blood.
 2. The blood substitute of claim 1, whereinsaid tetrameric hemoglobin has a molecular weight greater than 60,000daltons.
 3. The blood substitute of claim 1, wherein said bloodsubstitute is endotoxin and stroma free.
 4. The blood substitute ofclaim 1, wherein said blood substitute is non-pyrogenic.
 5. The bloodsubstitute of claim 1, wherein said blood substitute is deoxygenated. 6.The blood substitute of claim 1, wherein said tetrameric hemoglobincomprises four bovine polypeptides covalently cross-linked together withbis 3′,5′ dibromo salicyl fumarate.
 7. The blood substitute of claim 1,wherein said tetrameric hemoglobin comprises four bovine polypeptidescovalently cross-linked together with a poly-functional agent selectedfrom the group consisting of: glutaraldehyde; succindialdehyde;activated polyoxyethylene; activated dextran; α-hydroxy aldehyde;glycolaldehyde; N-maleimido-6-aminocaproyl-(2′-nitro,4′-sulfonicacid)-phenyl ester; m-maleimidobenzoic acid-N-hydroxysuccinimide ester;succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate;sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate;m-maleimidobenzoyl-N-hydroxysuccinimide ester;m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester;N-succinimidyl(4-iodoacetyl)aminobenzoate; sulfosuccinimidyl(4-iodoacetyl)aminobenzoate; succinimidyl 4-(p-maleimidophenyl)butyrate;sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate;1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;N,N′-phenylene dimaleimide; bis-imidate; acyl diazide; and aryldihalide.
 8. The blood substitute of claim 1, wherein thethiol-protecting group is a carboxymethyl such that the tetramerichemoglobin is a carboxymethylated tetrameric hemoglobin.
 9. The bloodsubstitute of claim 1, wherein the thiol-protecting is one of:4-pyridylmethyl; acetylaminomethyl; alkoxyalkyl; triphenylmethyl;carboxymethyl; acetyl; benzyl; benzoyl; tert-butoxycarbonyl;p-hydroxyphenacyl; p-acetoxybenzyl; p-methoxybenzyl; 2,4-dinitrophenyl;isobutoxymethyl; tetrahydropyranyl; acetamidomethyl; bezamidomethyl;bis-carboethoxyethyl; 2,2,2-trichloroethoxycarbonyl;tert-butoxycarbonyl; N-alkyl carbamate; and N-alkoxyalkyl carbamate. 10.The blood substitute of claim 1, in combination with an oxygenimpermeable polymer bag in which the blood substitute is enclosed.
 11. Ablood substitute suitable for administration to a human patient, theblood substitute comprising: tetrameric hemoglobin; and apharmaceutically acceptable carrier; wherein said tetrameric hemoglobincomprises four polypeptide chains derived from a non-human sourcecovalently cross-linked together; and wherein said tetrameric hemoglobinincludes at least one cysteine moiety chemically modified with athiol-protecting group such that said at least one cysteine moiety isincapable of binding nitric oxide; and wherein said covalentlycross-linked tetrameric hemoglobin has a p50 for oxygen greater thanthat of human whole blood.
 12. The blood substitute of claim 11, whereinsaid four polypeptide chains are bovine polypeptide chains.
 13. Theblood substitute of claim 11, wherein said four polypeptide chains areporcine polypeptide chains.
 14. The blood substitute of claim 11,wherein said blood substitute is deoxygenated and non-pyrogenic.
 15. Theblood substitute of claim 11, in combination with an oxygen impermeablepolymer bag in which the blood substitute is enclosed.
 16. The bloodsubstitute of claim 11, wherein said cross-linked tetrameric hemoglobincomprises hemoglobin proteins derived from a non-human source andcross-linked with bis 3′,5′ dibromo salicyl fumarate.
 17. The bloodsubstitute of claim 11, wherein said cross-linked tetrameric hemoglobincomprises hemoglobin proteins derived from a non-human source andcross-linked with a poly-functional agent selected from the groupconsisting of: glutaraldehyde; succindialdehyde; activatedpolyoxyethylene; activated dextran; α-hydroxy aldehyde; glycolaldehyde;N-maleimido-6-aminocaproyl-(2′-nitro,4′-sulfonic acid)-phenyl ester;m-maleimidobenzoic acid-N-hydroxysuccinimide ester; succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate; sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate;m-maleimidobenzoyl-N-hydroxysuccinimide ester;m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester;N-succinimidyl(4-iodoacetyl)aminobenzoate; sulfosuccinimidyl(4-iodoacetyl)aminobenzoate; succinimidyl 4-(p-maleimidophenyl)butyrate;sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate;1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;N,N′-phenylene dimaleimide; bis-imidate; acyl diazide; and aryldihalide.
 18. The blood substitute of claim 11, wherein thethiol-protecting group is a carboxymethyl such that the covalentlycross-linked tetrameric hemoglobin is a covalently cross-linkedcarboxymethylated tetrameric hemoglobin.
 19. The blood substitute ofclaim 11, wherein the thiol-protecting is one of: 4-pyridylmethyl;acetylaminomethyl; alkoxyalkyl; triphenylmethyl; carboxymethyl; acetyl;benzyl; benzoyl; tert-butoxycarbonyl; p-hydroxyphenacyl;p-acetoxybenzyl; p-methoxybenzyl; 2,4-dinitrophenyl; isobutoxymethyl;tetrahydropyranyl; acetamidomethyl; bezamidomethyl;bis-carboethoxyethyl; 2,2,2-trichloroethoxycarbonyl;tert-butoxycarbonyl; N-alkyl carbamate; and N-alkoxyalkyl carbamate. 20.A blood substitute suitable for administration to a human patient, theblood substitute comprising: tetrameric hemoglobin in a pharmaceuticallyacceptable carrier; wherein said tetrameric hemoglobin comprises fourpolypeptide chains derived from a non-human source covalentlycross-linked together with bis 3′,5′ dibromo salicyl fumarate; andwherein said tetrameric hemoglobin includes at least one cysteine moietychemically modified with a thiol-protecting group such that said atleast one cysteine moiety is incapable of binding nitric oxide; andwherein said tetrameric hemoglobin has a p50 for oxygen greater than 27mm Hg.